BfOLOOY 

LIBRARY 
G 


BIOCHEMISTKY   OF   MUSCLE  AND   NEKVE 


TEN    LECTURES 


ON 


BIOCHEMISTRY     OF 
MUSCLE  AND  NERVE 


BY  W.  D.  HALLIBURTON,  M.D.,  F.R.S. 

• ; 

PROFESSOR  OF  PHYSIOLOGY,   KING'S  COLLEGE,   LONDON 
EDITOR   OF   KIRKES'    "  HAND-BOOK   OF   PHYSIOLOGY  " 


WITH    ILLUSTRATIONS 


PHILADELPHIA 
P.  BLAKISTON'S   SON   AND   CO. 

1012    WALNUT    STREET 
1904 


10 


BIOLOGY 


:\A 


CRC 


PREFACE 


IN  the  early  part  of  1903  I  delivered  a  course  of  eight  lectures 
on  the  chemical  aspect  of  muscle  and  nerve  physiology  at  the 
Physiological  Laboratory  of  the  University  of  London.  A  little 
later  I  received  an  invitation  from  the  University  and  Bellevue 
Hospital  College,  New  York,  to  deliver  there  a  course  of  lectures 
on  some  subject  of  chemico-physiological  interest.  Dr  C.  A. 
Herter  had  for  some  years  been  in  the  habit  of  giving  a  course 
of  lectures  on  Chemical  Pathology  at  this  medical  school,  and 
when  he  received  the  appointment  of  Professor  of  Pharmacology 
at  the  Columbia  University,  New  York,  he  desired  to  perpetuate 
the  course  at  his  old  college.  This  he  did  by  a  generous  gift, 
which  enables  the  Bellevue  authorities  to  annually  invite  some 
one  who  has  particularly  worked  at  chemical  physiology  or 
pathology  to  deliver  such  a  course  of  lectures.  It  is  hardly 
necessary  for  me  to  say  that  I  felt  highly  honoured  at  the 
request,  that  I  should  be  the  first  Herter  Lecturer;  and  as  my 
lectures  at  the  University  of  London  had  not  been  published,  I 
determined  to  take  the  same  subject,  and  the  lectures,  twelve  in 
number,  were  given  in  New  York  during  January  of  the  present 
year.  The  little  book  which  I  now  present  is  the  outcome  of 
these  two  courses  of  lectures.  For  convenience  I  have  divided 
the  subject  into  ten  lectures,  the  actual  order  and  arrangement 
being  necessarily  somewhat  different  from  those  which  were 
actually  delivered.  In  New  York  the  longer  course  enabled  me 
to  amplify  some  of  the  subjects  which  I  had  to  pass  over  rapidly 
in  the  lectures  given  in  London.  The  arrangements  of  the  two 
laboratories  being  different,  I  had  also  to  vary  somewhat  the 


vi  PREFACE 

experiments  which  I  selected  to  illustrate  the  lectures ;  but  it 
has  obviously  become  necessary,  in  writing  them  out,  to  amalga- 
mate the  two  courses  so  as  to  make  them  read  as  one. 

At  the  University  of  London,  Dr  Waller,  and  in  New  York, 
Professor  Graham  Lusk,  placed  the  resources  of  their  labora- 
tories at  my  disposal,  and  it  is  my  pleasant  duty  to  thank  them 
for  the  assistance  they  so  ungrudgingly  gave  me. 

My  thanks  are  also  due  to  Dr  Alcock  and  to  Professor  T.  G. 
Brodie,  who  helped  me  with  my  experiments  in  London  ;  and  to 
Professor  Mandel,  Dr  Arthur  Mandel,  and  Dr  Wolf,  who  per- 
formed similar  kindly  offices  in  New  York. 

The  publication  of  this  book  gives  me  an  opportunity  of 
presenting  in  a  systematised  way  the  numerous  researches  on 
muscle  and  nerve  which  have  been  carried  out  in  my  laboratory 
during  the  past  few  years,  and  here  again  I  have  to  acknowledge 
with  gratitude  the  help  and  co-operation  of  colleagues,  friends, 
and  pupils  who  have  worked  with  me. 

I  append  to  this  preface  a  list  of  the  published  papers  which 
have  been  the  result  of  this  work,  and  which  have  formed  the 
basis  on  which  these  lectures  have  been  built. 


W.  D.  HALLIBURTON. 


KING'S  COLLEGE,  LONDON, 
1904. 


PRINCIPAL    PAPERS 


THE  following  list  gives  the  names  and  places  of  publication 
of  the  principal  papers  referred  to  in  the  following  lectures, 
which  have  been  carried  out,  either  by  myself,  or  in  conjunction 
with  colleagues,  or  under  my  superintendence.  The  first  six 
were  published  during  the  time  I  was  Assistant  Professor  of 
Physiology  at  University  College,  London ;  the  remainder 
were  published  since  I  have  held  the  Chair  of  Physiology  at 
King's  College,  London  : — 

1.  "An  arrangement  for  determining  the  Temperature  of  Heat  Coagulation  of 

Proteids,"  by  W.  D.  Halliburton,  Jour,  of  Phys.,  vol.  iv.,  1883. 

2.  "The  Proteids  of  Serum,"  by  W.  D.  Halliburton,  Ibid.,  vol.  v.,  1884. 

3.  "  On  Muscle  Plasma,"  by  W.  D.  Halliburton,  Ibid.,  vol.  viii. ;  Preliminary 

Communication  in  Proc.  Roy.  Soc.,  vol.  xlvii.,  1887. 

4.  "  Cerebro-spinal  Fluid,"  by  W.  D.  Halliburton,  Report  of  Spina  Bifida 

Committee,  Clin.  Soc.  Trans.,  vol.  xviii.,  1885. 

5.  "  Cerebro-spinal  Fluid,"  by  W.  D.  Halliburton,  Jour,  of  Phys.,  vol.  x.,  1889. 

6.  Report  on  Pathological  Effusions,  by  W.  D.  Halliburton,  Brit.  Med.Jour., 

1890. 

7.  "  Mucin   in   Myxoedema,   by  W.   D.   Halliburton,  Jour,   of  Path.,  May 

1892. 

8.  "  The  Chemical  Physiology  of  the  Animal  Cell,"  by  W.  D.  Halliburton, 

Goulstonian  Lectures  delivered  before  the  Royal  College  of  Physicians, 
London,  Brit.  Med.  Jour.,  nth,  i8th,  and  25th  March  1893. 

9.  "The  Fibres  of  Retiform  Tissue,"  by  R.  A.  Young,  B.Sc.,Jour.  of  Phys., 

vol.  xiii.,  1892. 

10.  "  On  Fractional  Heat  Coagulation,"  by  R.  T.  Hewlett,  M.D.,  Ibid.,  vol. 

xiii.,  1892. 

11.  "The  Proteids  of  Nervous  Tissue,"  by  W.  D.  Halliburton,  Ibid.,  vol.  xv., 

1893. 
vii 


viii  PRINCIPAL  PAPERS 

12.  "Note  on  the  Chemistry  of  Muscle,"  by  Arthur  Whitfield,  M.D.,  Jour. 

of  Phys.,  vol.  xvi.,  1894. 

13.  "  Chemistry  of  Connective  Tissue,"  by  R.  A.  Young,  M.D.,  Ibid.,  vol.  xvi., 

1894. 

14.  "  Nucleo-albumins  and    Intra vascular  Coagulation,"  by   W.   D.  Halli- 

burton and  T.  Gregor  Brodie,  M.D.,  Ibid.,  vol.  xvii.,  1894. 

15.  "  Nucleo-proteids,"  supplementary  paper,  by  W.  D.  Halliburton,  Ibid., 

vol.  xviii.,  1895. 

16.  "Creatinine  in  Blood,"  by  P.  C.  Colls,  Ibid.,  vol.  xx.,  1896. 

17.  "Hydrolysis  of  Glycogen,"  by  M.  Christine  Tebb,  Ibid.,  vol.  xxii.,  1897. 

1 8.  The  Articles   entitled  "The  Chemical  Constituents  of  the  Body  and 

Food,"  and  "  The  Chemistry  of  the  Tissues  and  Organs,"  in  Schafer's 
Text-Book  of  Physiology,  by  W.  D.  Halliburton,  vol.  i.,  1898. 

19.  "An  Intestinal  Plethysmograph,"  by  A.  Edmunds,  B  Sc.,  Jour,  of  Phys., 

vol.  xxii.,  1898. 

20.  "  Preliminary  accounts   of  the   Physiological   Action   of   Choline    and 

Neurine,"  by  T.  W.  Mott,  M.D.,  F.R.S.,  and  W.  D.  Halliburton,  Proc. 
Phys.  Soc.,  February  1897,  February  1898,  and  February  1899. 

21.  "The   Physiological  Action   of  Choline  and   Neurine,"  by   the    same 

authors.  Abstract  in  Proc.  Roy.  Soc.,  vol.  Ixv.,  1899;  full  paper  in 
Phil.  Trans,  of  the  Roy.  Soc.,  series  B,  vol.  cxci.,  1899. 

22.  "  Note  on  the  Blood  in  a  case  of  Beri-beri,"  by  the  same  authors,  Brit. 

Med.  Jour.,  28th  July  1899. 

23.  "  Observations  on  the  Cerebro-spinal  Fluid  in  the  Human  Subject,"  by 

St  Clair  Thomson,  M.D.,  L.  Hill,  M.B.,  and  W.  D.  Halliburton,  Proc. 
Roy.  Soc.,  vol.  Ixiv.,  1899. 

24.  The  Croonian   Lectures   on  the  Chemical  Side  of   Nervous   Activity, 

delivered  before  the  Royal  College  of  Physicians,  June  1901,  by 
W.  D.  Halliburton.  Abstracts  of  the  four  lectures  were  published 
in  the  Brit.  Med.  Jour.,  I5th  and  22nd  June  1901.  The  full  lectu/es 
were  published  as  a  separate  book  by  Bale,  Sons,  &  Daniellson, 
London,  1901. 

Dr  Mott's  Croonian  Lectures  on  the  Degeneration  of  the  Neurone, 
delivered  in  1900  (same  publishers),  also  contained  much  of  our 
joint  work. 

25.  "The  Chemistry  of  Nerve  Degeneration,"  by  F.  W.  Mott,  and  W.  D. 

Halliburton.  Abstract  published  in  the  Proc.  Roy.  Soc.,  vol.  Ixviii., 
1901  ;  full  paper  in  the  Phil.  Trans,  of  the  Roy.  Soc.,  series  B,  vol. 
cxciv.,  1901. 

26.  "  Regeneration   of  Nerves,"  by  the   same   authors,    Preliminary   Com- 

munication, Annual  Reports  of  the  British  Association,  Belfast, 
1902. 

27.  "  Regeneration  of  Nerves,"  by  F.  W.  Mott,  Arthur  Edmunds,  and  W.  D. 

Halliburton,  Second  Preliminary  Communication,  Proc.  Phys.  Soc., 
March  1904. 


PRINCIPAL  PAPERS  ix 

28-  "The  Proteids  which  may  occur  in  Urine,"  by  W.  D.  Halliburton,  Trans. 

Path.  Soc.,  London,  vol.  li.,  1900. 
29.  "  The  Physiological  Effects  of  Extracts  of  Nervous  Tissues,"  by  W.  D. 

Halliburton,  four,  of  Phys.,  vol  xxvi.,  1901. 
50.  "The  Veratrine-like  action  of  Glycerin,"  by  H.  Willoughby  Lyle,  M.D., 

Proc.  Phys.  Soc.,  January  1901. 

31.  "The  Action  of  Ether  and  Chloroform  in  the  Neurons  of  Rabbits  and 

Dogs/'  by  Hamilton  Wright,  M.D.,/our.  of  Phys.,  vol.  xxvi.,  1901. 

32.  "The   Action  of  Ether  and  Chloroform  on  the  Cerebral  and  Spinal 

Neurons  of  Dogs,"  supplementary  paper,  by  Hamilton  Wright,  Ibid., 
vol.  xxvi.,  1901. 

33.  "  Specific  Gravity  of  the  Brain,"    by  R.  H.  C.  Gompertz,  B.Sc.,  Ibid., 

xxvii.,  1902. 

34.  "  Reticulin  and  Collagen,"  by  M.  Christine  Tebb,  Ibid.,  vol.  xxvii.,  1902. 

35.  "Fatigue  in  Non-medullated  Nerves,"  by  T.  Gregor  Brodie,  and  W.  D. 

Halliburton,  Ibid.,  vol.  xxviii.,  1902. 

36.  "  The  Coagulation  Temperature  of  Cell-globulin  and  its  Relation   to 

Hyperpyrexia,"    by   F.   W.    Mott    and    W.    D.    Halliburton,    Mott's 
Archives  of  Neurology,  vol.  ii.,  1903. 

37.  "  The  Choline  Test  for  Active  Degeneration  of  the   Nervous  System," 

by  F.  W.  Mott,  Ibid.,  vol.  ii.,  1903. 

38.  "  Heat  Contraction  in   Nerve,"  Preliminary  Communication  ;  by  T.  G. 

Brodie  and  W.  D.  Halliburton,  Proc.  Phys.  Soc.,  July  1903. 

39.  "The  Precipitation  of  Proteids  by  Alcohol  and  other  Reagents,"  by 

M.  Christine  Tebb,  Jour,  of  Phys.,  vol.  xxx.,  1904. 

40.  "  Heat  Contraction  in   Nerve,"  by  T.  G.  Brodie  and  W.  D.  Halliburton, 

in  course  of  publication  in  the  Jour,  of  Phys. 


CONTENTS 


LECTURE  I 

PAO1! 

INTRODUCTORY — COMPOSITION  OF  MUSCLE.          v.    .    •    .  i 

Introductory — General  Composition  of  Muscular  Tissue — The 
Proteids  of  Muscle — Muscle  Plasma — Rigor  Mortis^  its 
Onset  and  Disappearance. 


LECTURE  II 

HEAT     RIGOR     OF     MUSCLE  —  EUGLOBULINS     AND     PSEUDO- 
GLOBULINS          ....... 

The  Steps  in  Heat  Rigor  ;  their  Coincidence  with  the  Coagula- 
tion Temperatures  of  the  Muscle  Proteids  ;  Differences 
between  Cold  and  Warm-blooded  Animals — Euglobulin 
and  Pseudo-Globulin  of  Blood  Serum,  Egg-White  and 
Muscle. 


LECTURE  III 

THE  PIGMENTS  OF  MUSCLE — PROPERTIES  OF  NUCLEO-PROTEIDS 

— THE  FERMENTS  OF  MUSCLE  ....         26 

Pale    and    Red    Muscles — Haemoglobin,    Myohasmatin,    Lipo- 
chromes  —  Properties   of    Nucleo-Proteids  —  Ferments    in 
Muscle ;    Myosin   Ferment,  Proteolytic    Ferment,   Amylo- 
lytic  Ferment,  Maltase,  Glycolytic  Ferment, 
xi 


xii  CONTENTS 

LECTURE  IV 

PAGB 

THE  EXTRACTIVES  AND  SALTS  OF  MUSCLE  ,  .          .         32 

List  of  Extractives— Carbohydrates,  Fat,  Lactic  Acids,  Nitro- 
genous Extractives,  Urea,  Creatine  and  Creatinine,  Purine 
Substances,  Carnic  Acid — Inorganic  Salts. 

LECTURE  V 

CHEMICAL    CHANGES    ACCOMPANYING    THE    CONTRACTION    OF 

MUSCLE — CHEMISTRY  OF  TENDON     ....         49 

Chemical  Tonus — Inogen  Theory — Comparison  of  Chemical 
Changes  which  occur  during  Contraction  and  on  Death  of 
Muscle — Reducing  Substances  in  Muscle — Chemistry  of 
Tendon  ;  Collagen,  Gluco-Proteids,  Reticulin. 


LECTURE  VI 
THE  CHEMICAL  COMPOSITION  OF  NERVOUS  TISSUES        .  .         57 

Introductory — Relation  of  Water  and  Solids — Specific  Gravity 
— Solids  of  Nervous  Tissues  ;  Proteids  ;  Phosphorised  Fats, 
Protagon,  Lecithin  and  its  Decomposition  Products, 
Kephalin — The  Cerebrins  or  Cerebrosides — The  Cerebro- 
Spinal  Fluid. 

LECTURE  VII 
METABOLISM  IN  NERVOUS  TISSUES  .  .          .          .  .         78 

Necessity  for  Oxygen — Reaction  of  Nervous  Tissues — The 
Hypothetical  Production  of  Carbon  Dioxide  during  Nervous 
Action — Evidence  of  Metabolic  Activity  in  Nervous  Struc- 
tures derived  from  the  Examination  of  Cerebro-Spinal 
Fluid,  and  of  Saline  Extracts  of  Nervous  Tissues,  and  from 
the  Histological  Examination  of  Nerve-Cells — Relative 
Importance  of  Central  and  Peripheral  Fatigue — Ergo- 
graphs — Nissl's  Granules  ;  Chromatolysis — The  Absence 
of  Fatigue  Changes  in  Nerve-Fibres,  Medullated  and  Non- 
medullated— Sleep  and  Narcosis. 


CONTENTS  xiii 

LECTURE  VIII 

PAGE 

THE  COAGULATION  TEMPERATURE  OF  THE  NERVE-PROTEIDS, 
AND  ITS  BEARING  ON  THE  QUESTIONS  OF  :  (i)  THE 
GALVANOMETRIC  RESPONSE  OF  NERVE  UNDER  VARYING 
TEMPERATURES  ;  (2)  HEAT  CONTRACTION  IN  NERVE  ; 
AND  (3)  HYPERPYREXIA  .  .  .  .  .  102 


LECTURE  IX 

THE     CHEMICAL     PATHOLOGY    OF     CERTAIN     DEGENERATIVE 

NERVOUS  DISEASES      .  .  .  .  .116 

Chemical  Pathology  of  General  Paralysis  of  the  Insane — Phy- 
siological Action  of  Choline  and  Neurine  —  Chemical 
Reactions  of  Choline  and  Neurine — Choline  in  the  Blood, 
and  Cerebro-Spinal  Fluid  in  Degenerative  Diseases  of 
the  Nervous  System,  Central  and  Peripheral. 


LECTURE  X 
DEGENERATION  AND  REGENERATION  OF  NERVES  .          ,          .        134 

Wallerian  Degeneration  ;  Disuse  Atrophy — Experiments  on 
Animals ;  Observations  on  their  Blood — Chemical  and 
Histological  Examination  of  Cut  Nerves — Chemical  Mean- 
ing of  the  Marchi  Reaction — Regeneration  of  Nerves — 
Conclusion. 


INDEX     .  .  .  .  .  .  .  .  .        153 


LIST     OF     ILLUSTRATIONS 


r.  Heat  Rigor  Curve  of  Mammalian  Muscle  .  .  .  .  15 

2.  „  „        Frog's  Muscle  .  .    '  .          18 

3.  Absorption  Spectra  of  Myohaematin          ~.-'          .   On  plate  to  face  p.  28 

4.  Tracing  of  Blood  Pressure,  showing  Effect  of  injecting  Extract 

of  Brain  ;  before  Atropine  V"         .  .  84 

5.  The  same  ;  after  Atropine  ....  .84 

6.  Four    Drawings,   illustrating  the   Methylene-blue    Reaction   of 

Nerve-Cells  .  .  .  '  .   On  plate  to  face  p  89 

7.  Outline  Drawing  of  Apparatus  for  obtaining  Splenic  Curves       .  92 

8.  Arrangement  of  the  Apparatus  in  Connection  with  the  Splenic 

Nerve        ........          93 

9    Tracings  of  Splenic  Contractions    .  .  .   On  plate  to  face  p.  94 

10.  Moniliform  Enlargements  on  Dendrons  produced  by  Anaesthesia          98 
n.  Galvanometer  applied  to  Nerve      .  .  .  .  .103 

12.  Effect  of  Hyperpyrexia  on  Nerve-Cells  .  On  plate  to  face  p.  114 

13  Tracing  to  illustrate  Effect  of  Choline  on  Blood  Pressure  and 

Intestinal  Volume  .  .  .  .  .  .120 

14.  Tracing  to  illustrate  Effect  of  Neurine  on  Blood  Pressure  and 

Respiration         .  .  .  .  .  .  .  .122 

15.  Crystals  of  Platino-chloride  of  Choline       .  .  .  .124 

1 6.  The  same  Crystals  prepared  from  Pathological  Cerebro-Spinal 

Fluid     |<  .-.-..  .  .  .  .  .125 

17.  Nerve  Degeneration  in  Alcoholic  Neuritis  .  .  .129 

18.  Tracing  to  illustrate  Effect  of  Choline  (prepared  from  Patho- 

logical  Blood)  on   Blood  Pressure  and  Respiration  ;  before 
Atropine   .  .  .  .  .  .  .  .130 

19.  The  same  ;  after  Atropine  .  .  .  .  .  .131 

20.  Tracing  of  Blood  Pressure  to  illustrate  the  Effect  of  Choline 

obtained  from  the  Blood  of  a  Cat,  after  Section  of  both  Sciatic 
Nerves;  before  Atropine  .  .  .  .  135 

21.  The  same ;  after  Atropine   .  .  .  .  .  .136 


xvi  LIST  OF  ILLUSTRATIONS 

FIG. 

22.  Transverse  Section  of  Cat's  Nerve,  53  hours  after  Section. 

23.  „  „  „  3  days  after  Operation. 

24.  „  .,  „  8  days  after  Operation. 

On  plate  to  face  p.  \i 

25.  Longitudinal  Section  of  Nerve,  99  hours  after  Operation. 

26.  ,,  „  „         10  days  after  Operation. 

27.  „  „  „         27  days  after  Operation. 

On  plate  to  face  p.  n\ 

28.  Single  Fibre  from  Nerve  8  days  after  Section,  to  show  Division 

of  Neurilemmal  Nucleus. 

29.  High-power  View  of  Fig.  27,  to  show  the  Part  played  by  Phago- 

cytes and  Neurilemmal  Cells  in  removing  Degenerated  Fat. 

30.  Longitudinal  Section  of  Regenerated  Nerve. 

31.  Transverse  Section  of  the  same. 

On  plate  to  face  p.  14 

32.  Longitudinal  View  of  Nerve  44  days  after  Operation,  showing 

Elongated  Neurilemmal  Cells. 

33.  Transverse  Section  of  Regenerated  Nerve,  stained  to  show  Axis- 

Cylinder. 

On  plate  to  face  p.  14 


BIOCHEMISTRY   OF    MUSCLE 
AND    NERVE 


LECTURE    I 

INTRODUCTORY.      COMPOSITION    OF   MUSCLE 

THE  rapid  growth  of  the  science  of  Physiology  has  been 
accompanied  by  specialisation  on  the  part  of  its  disciples.  This 
has  become  necessary  because  it  is  impossible  for  any  one 
person  to  follow  into  its  many  ramifications  all  the  different 
branches  into  which  Physiology  is  now  subdivided. 

One  outward  and  visible  sign  of  this  inward  manner  of 
growth  is  the  institution  of  lectures  such  as  those  I  have  the 
honour  of  addressing  to  you  now.  This  comparatively  modern 
departure  is,  in  my  opinion,  of  benefit  both  to  students  and  to 
teachers.  It  enables  the  taught  to  hear,  from  an  investigator 
at  first  hand,  the  results  of  his  researches,  and  to  see  the  main 
experiments  upon  which  his  results  depend.  It  gives  a  new 
inspiration  to  the  lecturer  to  speak  to  a  new  class,  and  to  find 
himself  in  the  surroundings  of  laboratories  other  than  his  own. 

Among  the  special  subjects  of  which  various  physiologists 
have  made  a  life-study,  that  of  muscle  and  nerve  is  one  that 
has  always  found  numerous  adherents.  The  two  tissues  are  so 
important,  and  so  closely  associated,  that  this  is  not  surprising. 
In  some  hands,  the  mechanical,  in  others,  the  electrical  side  of 
this  attractive  subject  has  been  taken  up.  The  chemical  side, 
however,  is  the  one  which  has  always  most  interested  me,  and  I 
hope  soon  to  interest  you  in  it  also.  A  paper  on  the  chemistry 

A 


a  INTRODUCTORY  [LECT. 

of  muscle  was  the  result  of  one  of  my  earliest  researches  some 
years  ago  ;  recently,  the  chemistry  of  nerve  has  occupied  me  and 
the  workers  in  my  laboratory.  I  want  to  try  and  weave  these 
various  investigations  together,  and  present  you  with  a  con- 
nected account  of  the  whole. 

The  specialisation  to  which  I  just  referred  in  speaking  of 
the  growth  of  Physiology  is  an  evil.  It  tends  to  narrow  the 
outlook  of  the  investigator.  The  ultra-specialist  is  apt  to 
confine  himself  so  closely  to  his  own  groove  that  he  forgets  to 
notice  what  is  occurring  in  the  parallel  and  inter-crossing 
grooves  of  others.  But  those  who  devote  themselves  to  the 
chemical  side  of  Physiology  run  but  little  danger  of  this  kind. 
The  subject  cannot  be  studied  apart  from  other  branches  of 
Physiology,  so  closely  are  both  roots  and  branches  intertwined. 
I  have  entitled  these  lectures  the  Biochemistry  of  Muscle  and 
Nerve,  because  the  work  was  started  from  the  chemical  point  of 
view.  But  as  it  progressed,  it  grew  into  some  channels  which 
are  histological,  others  pharmacological,  others  still  of  a  purely 
experimental  kind  ;  and  in  all  cases  the  strictly  physiological 
runs  so  imperceptibly  into  the  pathological,  that  any  hard-and- 
fast  line  between  the  two  is  impossible  to  draw.  It  will  be  my 
duty,  therefore,  to  direct  your  attention,  later  on,  to  subjects 
which  are  not  chemical  at  all,  or  only  incidentally  so. 

Physiology  is  still  in  some  of  the  Scotch  universities  called 
by  its  old  name,  the  "  Institutes  of  Medicine."  This  title 
embodies  an  important  truth.  A  medical  student  should  not 
study  Physiology  merely  to  pass  a  certain  examination,  and 
then  forget  all  about  it.  It  is  the  substratum  on  which  he  must 
subsequently  build  his  knowledge  of  Pathology  and  Medicine. 
I  shall  endeavour  to  bear  this  in  mind  myself,  and  point  out, 
as  I  go  along,  the  pathological  and  practical  bearing  of  the 
subjects  I  bring  before  you. 

I  must,  however,  spend  no  more  time  on  general  reflections 
such  as  these.  Our  time  will  be  fully  occupied  in  the  more 
serious  work  before  us,  and  I  will  therefore  at  once  plunge  into 
the  middle  of  things,  and  ask  you  to  consider  with  me,  for  the 
remainder  of  the  hour,  the  general  composition  of  the  first  of 
the  two  tissues  we  are  to  study,  namely,  Muscle. 


I.]  COMPOSITION  OF  MUSCLE  3 

General  Composition  of  Muscular  Tissue 

Muscle  is  made  up  of  a  number  of  thread-like  structures 
called  muscular  fibres,  and  is  subdivided  into  varieties  accord- 
ing to  the  structure  of  these  fibres.  Some  are  transversely 
striated,  others  not  This  histological  classification  corresponds 
very  closely  to  the  physiological  subdivision  of  the  muscles 
into  voluntary  and  involuntary  respectively  ;  the  most  important 
exception  being  cardiac  muscle,  which,  though  transversely 
striated,  is  nevertheless  involuntary. 

By  far  the  greatest  amount  of  work  in  muscular  chemistry 
has  been  performed  upon  the  voluntary  muscles,  and  it  is  the 
composition  of  this  variety  that  I  will  ask  you  first  to  consider. 

Muscle,  as  usually  obtained,  is  mixed  with  a  certain  amount 
of  investing  connective  tissue,  and  the  gelatin  and  most  of  the 
fat  which  are  mentioned  in  tables  of  analyses  are  derived  from 
this. 

I  do  not  want  unduly  to  burden  you  with  figures,  plenty  of 
which  you  will  find  in  your  text-books.  It  will  be  sufficient  at 
this  stage  to  remind  you  that  muscular  tissue  contains  in  round 
numbers  25  per  cent,  of  solids,  and  the  remaining  75  per  cent, 
is  water. 

The  organic  substances  contained  in  the  solids  are  fairly 
numerous,  but  the  most  important  and  abundant  are  those 
of  proteid  nature.  Roughly,  20  out  of  the  25  parts  of 
solid  substance  are  proteid.  Although  it  is  not  my  intention 
to  deal  with  questions  of  dietetics,  I  may  mention  in  passing 
that  flesh  is  the  most  commonly  employed  source  of  our 
nitrogenous  food,  because  it  contains  proteid  both  in  abun- 
dance and  in  a  readily  digestible  form. 

The  remaining  5  per  cent,  of  the  solids  consist  of  numerous 
organic  materials  conveniently  grouped  together  as  extractives, 
and  a  certain  amount  of  inorganic  salts. 

The  Proteids  of  Muscle 

I  shall  take  first  the  proteids  of  muscle,  on  account  of  their 
preponderance  both  in  amount  and  in  importance.  Each 
muscular  fibre  consists  of  two  parts,  a  nucleated  sheath  or 


4  COMPOSITION  OF  MUSCLE  [LECT. 

sarcolemma  and  the  contractile  substance  which  it  encloses. 
The  sarcolemma  is  made  of  a  material  which  resembles 
elastin  in  its  solubilities,  and  the  nucleo-proteid,  which  we 
shall  deal  with  later,  is  doubtless  derived  from  the  nuclei. 
The  main  amount  of  proteid,  however,  is  contained  in  the  semi- 
fluid, contractile  substance.  By  means  of  a  press,  a  juice  can 
be  squeezed  out  of  perfectly  fresh  muscles,  and  this  was  termed 
by  Kiihne  the  muscle  plasma.  Like  blood  plasma,  this  coagulates, 
and  the  proteid  clot  is  called  myosin ;  when  this  occurs  within 
the  body  after  death,  the  stiffening  of  the  muscles  known  as 
rigor  mortis  is  the  result. 

Living  muscle  is  alkaline  in  reaction,  but  after  extreme 
activity  and  also  after  death,  the  reaction  becomes  acid ;  this  is 
in  part  due  to  the  development  of  sarcolactic  acid. 

Our  knowledge  of  the  proteids  of  muscle  dates  from  the 
investigations  of  Kiihne,  who  was  the  first  to  study  muscle 
plasma  with  profitable  results.  He  used  frog's  muscle,  which, 
after  having  been  freed  from  blood  and  then  frozen,  was  subjected 
to  strong  pressure.  The  expressed  juice  was  found  to  be  of 
syrupy  consistency,  and  alkaline  in  reaction.  After  lapse  of  time, 
especially  if  the  plasma  is  raised  to  the  temperature  of  the  air, 
it  clots,  arid  the  myosin  so  formed  contracts  to  a  slight  extent, 
squeezing  out  a  liquid  residue  «called  muscle  serum.  Kiihne 
found  this  latter  fluid  to  contain  a  proteid  coagulating  at  45°  C., 
an  alkali-albumin,  and  an  albumin,  with  salts  and  extractives 
in  addition.  These  investigations  date  a  great  many  years 
back  (1864) — that  is,  to  a  time  when  our  knowledge  of  the 
proteids  was  much  less  than  it  is  at  present ;  there  is  no  doubt 
that  the  natural  alkali-albumins  described  by  older  workers  are 
really  globulins. 

A  good  many  years  later,  I  was  successful  in  repeating  these 
experiments  with  mammalian  muscle,  and  was  able  to  show 
that,  not  only  does  cold  prevent  the  coagulation  of  muscle 
plasma,  but,  as  in  che  case  of  blood  plasma,  admixture  with 
solutions  of  neutral  salts  has  the  same  effect.  Addition  of 
water  to  the  salted  muscle  plasma  brings  about  coagulation 
(an  acid  reaction  making  its  appearance  simultaneously),  and 
this  occurs  more  rapidly  if  a  solution  of  "  myosin  ferment "  is 


I.]  MUSCLE  PLASMA  5 

added.  I  prepared  the  myosin  ferment  from  muscle  in  the 
same  way  that  Schmidt  prepared  fibrin  ferment  from  blood 
serum. 

Similar  saline  extracts  of  muscle  which  had  undergone  rigor 
mortis  resemble  salted  muscle  plasma  very  closely  ;  after  dilution 
they  undergo  coagulation ;  this  at  one  time  I  regarded  as  a 
recoagulation  of  the  redissolved  myosin,  and  the  process  is 
accompanied  as  before  with  increase  of  acidity.  Some  observers 
did  not  consider  this  to  be  a  true  coagulation,  but  merely  a 
simple  precipitation  of  myosin  by  dilution  with  water.  This 
may  be  true  in  part,  but  I  am  now  inclined  to  look  upon  the 
phenomenon  not  as  a  recoagulation  of  myosin  which  had 
already  clotted,  but  as  a  true  coagulation  (or  myosin  forma- 
tion) from  the  still  uncoagulated  residue  of  myosin-precursors. 
There  can,  I  think,  be  little  doubt  that  the  muscles  taken  were 
not  completely,  but  only  partially  in  the  state  of  rigor  mortis. 

The  analogy  of  muscle-clotting  to  blood-clotting  has  since 
Kiihne's  day  been  the  idea  underlying  most  of  the  investigations 
on  this  subject,  and  therefore  the  nomenclature  has  been  similar. 
Just  as  the  precursor  of  fibrin  in  the  blood  is  called  fibrinogen, 
so  the  precursor  of  myosin  in  the  muscles  has  been  termed 
myosinogen. 

We  have  already  seen  that  in  both  cases  cold  and  neutral 
salts  will  delay  clotting.  In  blood-clotting  it  is  well  known 
that  calcium  salts  are  essential,  and  there  are  some  facts,* 
though  they  are  not  yet  fully  established,  which  indicate  that 
this  element  is  also  important  in  the  process  of  muscle- 
coagulation.  The  probability  also  that  in  both  cases  one  has 
to  deal  with  the  action  of  a  ferment  has  also  been  mentioned. 

In  a  recent  paper,  however,  O.  Folinf  questions  the  coagulation  theory 
of  rigor  mortis  by  the  following  observation.  He  subjects  frogs'  muscles 
to  a  temperature  of  -15°  C.,  and  this  renders  them  stiff,  and  irresponsive  to 
stimuli.  From  these  muscles  he  finds  he  can  prepare  muscle  plasma  in  the 
usual  way,  which  has  all  the  characters  of  ordinary  muscle  plasma.  That 

*  Howell  and  Eaton,  Jour,  of  Phys.,  vol.  xiv.,  p.  219  ;  S.  Locke,  ibid.)  vol. 
xv.,  p.  1 19  ;  W.  H.  Howell,  ibid.,  vol.  xvi.,  476  ;  Cavazanni,  Archives  italiennes 
de  Biologie,  vol.  xviii.,  p.  156. 

f  American  Jour,  of  Phys.,  vol.  ix.,  p.  374. 


6  COMPOSITION  OF  MUSCLE  [LECT. 

is  to  say,  muscles  which  he  considers  have  undergone  true  rigor  mortis 
nevertheless  yield  muscle  plasma  which  subsequently  coagulates.  The  con- 
clusion I  should  draw  from  this  work,  is  not  that  the  coagulation  theory  is 
disproved,  but  simply  that  cold  rigor  is  not  true  rigor  mortis  at  all.  This 
view  is,  as  a  matter  of  fact,  supported  by  some  of  Folin's  own  observations ; 
for  instance,  there  is  no  formation  of  acid,  and  the  muscles,  though  stiff,  are 
still  perfectly  transparent. 

Let  me  now  show  you  one  or  two  simple  but  fundamental 
experiments.  The  rabbit  before  you  has  just  been  killed.  I 
open  the  abdomen  rapidly  and  insert  a  cannula  into  the  aorta, 
which  enables  me  to  wash  out  with  a  stream  of  saline  solution 
all  the  blood  from  the  muscles  of  the  lower  limbs.  This  being 
completed,  I  remove  the  skin,  chop  off  the  muscles,  and  mince 
them  finely  with  a  mincing  machine.  The  minced  muscle  is 
now  ground  up  in  a  mortar  with  clean  sand,  and  5  p^r  cent, 
solution  of  magnesium  sulphate,  and  the  salted  muscle  plasma 
so  obtained  is  filtered  off.  The  filtrate  is  viscid,  and  so  comes 
through  rather  slowly,  but  quite  a  small  quantity  is  all  we 
want  for  the  next  observation.  I  will  take  the  few  c.c.  that 
are  now  ready,  and  test  the  reaction  with  litmus,  and  you  see 
it  is  still  alkaline.  I  now  dilute  it  with  three  or  four  times  the 
quantity  of  water,  and  to  hurry  up  the  coagulation,  place  it  in 
the  water-bath  at  35°  C.  By  the  end  of  the  lecture,  I  expect 
to  be  able  to  show  you  a  typical  clot  of  myosin,  which  will 
subsequently  contract  and  squeeze  out  a  salted  muscle  serum.* 

Let  me  now  show  you  with  this  other  freshly  killed  rabbit 
another  way  of  making  muscle  plasma,  which  is  the  method 
more  recently  employed  by  v.  Fiirth.  I  obtain  the  minced, 
blood-free  muscle  as  before,  and  grind  it  up  this  time  with  a 
little  physiological  salt  solution,  and  some  clean  sand.  I  now 
wrap  successive  portions  in  pieces  of  muslin,  and  place  them  in 
a  press.  The  iron  lemon-squeezer  I  hold  is  very  effective  for 
this  purpose.  You  see  the  drops  of  muscle  plasma  (diluted,  of 
course,  to  a  slight  extent  with  the  salt  solution  added)  as  they 

*  This  expectation  was  not  fulfilled  ;  the  coagulation  did  not  take  place 
until  after  the  lecture  was  concluded.  The  specimen  was,  however,  kept 
and  exhibited  to  the  class  at  the  next  lecture.  The  fluid  was  then  distinctly 
acid  to  litmus,  and  by  means  of  Uffelmann's  colour  reaction,  the  acid 
present  was  shown  to  be  sarcolactic  acid. 


i.]  PROTE1DS  OF  MUSCLE  7 

come  through.  The  muscle  plasma  is  alkaline,  viscid,  and 
rather  opalescent  as  before,  and  filters  slowly.  Let  me,  how- 
ever, with  the  amount  we  have  now  obtained,  demonstrate 
rapidly  the  process  of  fractional  heat  coagulation,  a  very  useful^ 
method  as  a  preliminary  indication  of  the  existence  of  more 
than  one  proteid  in  a  solution.  I  place  a  thermometer  in  the 
test-tube,  and  the  test-tube  in  this  flask  which  is  filled  with 
water  at  about  30°  C,  and  the  temperature  of  which  is  slowly 
rising  owing  to  the  small  Bunsen  flame  beneath  it.  You  notice 
no  change,  but  we  must  watch  it  carefully  as  the  temperature 
rises.  The  temperature  is  now  42°  C.,  and  the  opalescence  is 
distinctly  deeper ;  it  is  now  47°,  and  the  deepening  opalescence 
has  culminated  in  the  deposition  of  flocculi  of  coagulated 
proteid  ;  we  must  keep  the  temperature  constant  at  47°  C.  for 
a  short  time,  in  order  to  ensure  that  all  the  proteid  which 
coagulates  at  this  temperature  has  separated  out.  Now  we 
filter  it  off,  and  you  notice  that  the  filtrate  is  perfectly  clear. 
We  place  the  filtrate  in  the  test-tube,  and  place  the 
test-tube  in  the  water-bath  again.  When  we  reach  47°  no 
further  coagulation  occurs,  so  we  continue  the  heating ;  now  a 
second  crop  of  flocculi  even  more  abundant  than  before  separates 
out,  and  the  thermometer  reads  56°  C.  Those  of  you  nearest 
the  table  will  have  noticed  that  the  coagulation  with  definite 
flocculi  was  preceded  as  before  with  a  gradually  deepening 
opalescence  a  degree  or  two  lower  than  56°. 

Another  fundamental  method  for  separating  proteids  from 
one  another,  is  that  which  is  known  as  salting  out.  You  will  be 
familiar  with  the  fact  that  globulins  are  more  readily  salted  * 
out  than  albumins.  Thus  half  saturation  with  ammonium 
sulphate  (one  of  the  most  frequently  employed  of  these  neutral 
salts  for  the  fractional  precipitation  of  proteids)  will  precipitate 
globulins  ;  complete  saturation  with  this  salt  is  necessary  to  pre- 
cipitate albumins.  There  is  no  doubt  that  of  the  two  proteids 
we  have  detected  by  fractional  heat  coagulation,  the  one  which 
coagulates  at  the  lower  temperature  is  salted  out  more  readily ; 
both,  however,  are  entirely  precipitated  by  half  saturation 
with  ammonium  sulphate,  and  so  may  provisionally  be  classed 
with  the  globulins.  The  small  amount  of  albumin  left  in 


8  COMPOSITION  OF  MUSCLE  [LECT. 

the  filtrate  probably  is  derived  from  adherent  blood  and 
lymph. 

V.  Fiirth*  states  he  has  been  able  to  completely  separate  the 
two  proteids  by  the  fractional  ammonium  sulphate  method.  I 
have  repeated  his  experiments,  and  have  not  succeeded  ;  with  a 
comparatively  small  amount  of  salt,  the  precipitate  consists 
mainly  of  the  proteid  which  enters  into  the  condition  of  a  heat 
coagulum  at  47°  C,  but  there  is  always  some  of  the  other  one  as 
well.  On  filtering  this  off  and  adding  more  salt,  the  bulk  of  the 
precipitate  is  the  56°  proteid,  but  mixed  with  it  is  the  remainder 
of  the  first  proteid.  In  other  words,  the  two  proteids  overlap 
in  relation  to  the  amount  of  salt  added. 

Now  these  two  proteids  are  the  precursors  of  myosin,  and  so 
may  be  included  in  the  general  term  myosinogen.  The  one 
which  coagulates  at  the  lower  temperature  is  less  important 
quantitatively,  and  so  I  called  it  para-myosinogen,  reserving  the 
name  myosinogen  for  its  more  abundant  neighbour.  Quantita- 
tively, it  has  been  found  that  the  relationship  between  the  two 
is  about  I  to  4  in  mammalian  voluntary  muscle. 

The  question  now  arises,  Are  these  two  proteids  really  globu- 
lins ?  There  is  no  doubt  that  para-myosinogen  is  a  globulin,  for 
not  only  is  it  readily  precipitable  by  neutral  salts,  but  it  is  also 
insoluble  in  water,  and  is,  therefore,  precipitated  by  dialysing 
away .  the  salts  that  enable  it  to  pass  into  solution.  It  is 
analogous  to  the  cell-globulin,  which  is  found  in  saline  extracts 
of  all  protoplasmic  structures.  Myosinogen,  on  the  other  hand, 
is  not  a  typical  globulin,  for  it  is  not  precipitated  by  dialysis, 
that  is  to  say,  it  is  soluble  in  water. 

Are  there  any  other  proteids  besides  these  two  in  muscle 
plasma  or  saline  extracts  of  muscle?  In  my  first  paper  in  1887, 
I  stated  there  were  three  others,  but  all  of  these  are  present  in 
minute  quantities.  These  are  a  globulin,  which  is  coagulated 
by  heat  at  a  temperature  higher  than  the  two  principal  pro- 
teids, and  an  albumin.  These,  however,  are  probably  due  to 
contamination,  with  small  amounts  of  blood  and  lymph.  The 
more  thoroughly  the  blood  and  lymph  are  washed  away,  the 

*  A  good  general  account  of  v.  Fiirth's  work  is  given  in  his  article  in  the 
Ergebuisse  der  Physiologic,  vol.  i.,  part  i,  1902,  p.  no. 


i.]  INVOLUNTARY  MUSCLE  9 

less  abundant  they  are.  The  third  of  these  extra  proteids  I 
named  myo-albumose,  but  I  soon  discovered  that  there  is  no 
albumose  in  muscle,  and  Dr  Whitfield,  in  my  laboratory,  con- 
clusively proved  this  to  be  so  with  the  new  methods  for  detect- 
ing albumoses,  which  were  available  at  the  time  he  worked. 

In  the  red  voluntary  muscles  there  is  a  small  quantity  of 
haemoglobin,  which  we  shall  deal  with  when  speaking  of  the 
pigments  of  muscle ;  and  in  all  muscles  there  is  a  small 
amount  of  nucleo-proteid. 

In  the  involuntary  muscles,  the  phenomena  of  rigor  are  much 
the  same  as  in  voluntary  muscle.  Swale  Vincent,*  who  is  the 
principal  worker  on  this  side  of  the  subject,  has  also  shown  that 
the  two  main  proteids  are  identical.  The  most  striking  differ- 
ence between  the  two  classes  of  muscle  lies  in  the  amount  of 
nucleo-proteid  present.  It  is  more  abundant  in  plain  muscle 
than  in  cardiac  muscle,  and  least  abundant  of  all  in  voluntary 
muscle.  In  other  words,  those  varieties  of  muscle  which  in 
process  of  development  have  departed  most  from  the  structure 
of  the  simple  animal  cells  from  which  all  muscular  fibres  ulti- 
mately develop,  are  those  which  possess  least  of  the  proteid, 
which  is  most  typical  of  simple  protoplasmic  cells. 

This  may  be  very  strikingly  shown  by  the  experiment 
I  am  next  going  to  perform.  I  have  made  an  extract  with 
a  0.15  per  cent,  solution  of  sodium  carbonate  of  equal  quan- 
tities of  the  three  varieties  of  muscle.  I  add  dilute  acetic 
acid  to  the  extract  of  plain  muscle,  and  obtain  an  abundant 
precipitate  of  nucleo-proteid  ;  I  do  the  same  with  the  extract  of 
cardiac  muscle,  and  the  precipitate  is  much  less  abundant,  while 
with  the  extract  of  voluntary  muscle,  the  precipitate  is  so  scanty 
that  those  at  the  back  of  the  room  must  be  content  with  my 
word  that  there  is  a  cloudiness  produced. 

But  now  we  must  return  to  our  two  principal  proteids,  and 
discuss  the  way  in  which  they  pass  with  the  coagulated  condition. 
Stewart  and  Sollmannf  hold  the  view  that  the  distinction 
between  the  two  proteids  is  mainly  artificial,  and  that  there  is 

*  Zeit.  f.  physiol.    Chem.,   1902,  vol.    xxxiv.,  p.  417.     Velichi   has   made 
similar  observations. 

t  Jour,  of  Phys.)  1899,  vol.  xxiv.,  p.  427. 


io  COMPOSITION  OF  MUSCLE  [LECT. 

really  only  one  percursor  of  the  clot  of  myosin.  But  although 
the  two  may,  in  a  sense,  be  integral  parts  of  a  complex  proteid, 
I  believe  with  v.  Fiirth  that  the  distinction  between  them  is  by 
no  means  artificial,  for  the  differences  between  them  are  many 
and  striking  ;  and  Brodie's  work  on  heat  rigor,  to  which  I  shall 
have  to  direct  your  attention  at  my  next  lecture,  conclusively 
proves  that  they  exist  as  separate  entities  in  the  intact  muscle. 
Whether  the  transformation  is  due  to  ferment  action  or  not, 
must  also  be  considered  in  the  light  of  v.  Fiirth's  recent 
unsuccessful  search  for  the  enzyme  as  unproven.  V.  Fiirth  and  I, 
however,  are  in  substantial  agreement ;  the  main  difference 
between  us  is  a  difference  of  terms.  V.  Fiirth's  names  for 
the  proteids  have  the  merit  of  brevity,  but  the  introduction 
of  new  words  for  things  which  have  previously  been  known 
by  other  names  is  always  confusing  to  the  student.  Para- 
myosinogen  passes  directly  into  the  clotted  condition  of 
myosin  or  muscle-fibrin  ;  but  myosinogen  first  passes  into  a 
•soluble  condition  (coagulable  by  heat  at  the  remarkably  low 
temperature  of  40°  C.)  before  it  clots  ;  this  soluble  stage,  which 
I  had  noted  in  my  own  work,  though  I  failed  to  give  a  correct 
interpretation  to  it,  may  be  called  soluble  myosin  (v.  Fiirth's 
soluble  myogen-fibrin).  We  may  put  this  in  a  diagrammatic 
way  as  follows  :— 

Proteids  of  the  living  muscle. 


I  I 

3sinogen.  Myosinogen. 

(Myosin  of  v.  Fiirth.)  (Myogen  of  v.  P'iirth.) 

Soluble  myosin. 


Myosin. 

(The  proteid  of  the  muscle  clot.) 

V.  Fiirth,  in  addition  to  his  work  on  mammalian  muscle,  has 
made  some  interesting  comparative  observations  on  the 
voluntary  muscles  of  cold-blooded  animals. 

He  found  that  in  these  (working  chiefly  with  frog's  muscle) 
that  the  soluble  myogen-fibrin  just  alluded  to  as  a  stage  in 
the  process  of  rigor  mortis  in  mammalian  muscle,  is  present  as 


I .  ]  COM  PA  RA  TI VE  C HEM  IS  TRY  OF  MUSCLE  1 1 

such  in  the  living  fresh  muscle  to  a  certain  extent.  He  dis- 
covered also  that  in  the  muscle  plasma  of  fishes  there  is  another 
peculiar  proteid,  which  he  called  myoproteid.  It  is  precipitable 
by  dialysis  and  by  acetic  acid,  but  is  not  coagulable  by  heat. 

This  work  has  been  extended  by  Hans  Przibram,*  who  has 
attempted  to  classify  the  animal  kingdom  on  the  basis  of  the 
muscle  proteids.  As  his  conclusions  are  based  on  the  examina- 
tion of  only  thirty  species  of  animals,  they  may  require  revision 
in  the  future,  but  such  as  they  are,  they  are  as  follow : — 

INVERTEBRATES. — Para-myosinogen  present  ;  myosinogen  absent. 
VERTEBRATES. — Para-myosinogen  and  myosinogen  both  present. 

Fishes :  In  addition  to  these  two  principal  proteids,  myoproteid  and 

soluble  myogen-fibrin  occur. 
Amphibia :   Like  fishes,  except  that  myoproteid  is  present   only  in 

traces. 

n>  j     '       I  Myoproteid    absent,    and    soluble    myogen-fibrin    only 
Mammals   \        present  when  rigor  mortis  commences. 

It  is  obviously  important  that  such  being  the  condition  of 
things  in  the  normal  state,  we  should  be  able  to  say  what  occurs 
in  the  various  pathological  conditions  of  which  muscle  may  be 
the  subject.  But  here,  as  is  only  to  be  expected  in  a  branch  of 
science  so  much  in  its  infancy,  very  little  has  as  yet  been  done. 
Indeed,  the  only  research  with  which  I  am  acquainted  is  one  by 
Steyrer.-f  He  states  that  on  prolonged  tetanisation  of  rabbit's 
muscle  the  amount  of  para-myosinogen  diminishes,  that  when 
degeneration  occurs  after  the  motor  nerves  are  cut  the  amount 
of  this  proteid  increases,  and  that  after  the  tendon  of  a  muscle 
has  been  cut  there  is  no  change  in  the  proportion  of  the  two 
proteids.  These  investigations  open  the  door  to  what  may 
prove  to  be  a  most  profitable  line  of  research,  and  work  such  as 
this  may  result  in  a  more  accurate  knowledge  of  the  various 
degenerative  processes  which  are  so  frequently  seen  in  muscle 
after  death  from  various  causes. 

A  few  more  words  on  rigor  mortis  will  conclude  all  I  shall 
trouble  you  with  to-day.  The  onset  of  post-mortem  rigidity  is 

*  Beitrdge  chem.  Phys.  n.  Path  ,  [902,  vol.  ii.,  p.  143. 
t  Ibid.,  1903,  vol.  iv.,  p.  234. 


12  COMPOSITION  OF  MUSCLE  [LECT.  i. 

not  the  only  problem  connected  with  that  subject ;  *  for  after  a 
varying  interval  the  rigor  passes  off  and  the  muscles  once  more 
relax  ;  what  is  the  cause  of  the  disappearance  of  rigor  ?  The 
usual  explanation  is  that  this  is  due  to  the  onset  of  putrefactive 
changes,  but  the  stiffening  sometimes  passes  off  too  quickly  to 
be  attributable  to  this  cause,  and  I  still  hold  to  the  opinion  that 
the  relaxation  is  due  to  the  action  of  an  enzyme  which  produces 
-an  autodigestion,  and  thus  a  softening  of  the  hardened  tissue. 
There  is  no  longer  any  reason  to  suppose  that  the  ferment  at 
work  is  pepsin  which  had  been  previously  absorbed  from  the 
alimentary  canal,  for  Hedinf  has  shown  that  the  proteolytic 
ferment  which  is  present  in  muscle,  as  in  many  other  animal 
tissues  (spleen,  kidney,  etc.),  is  more  like  trypsin  than  pepsin  in 
its  mode  of  action.  These  ferments,  however,  are  not  trypsin, 
for  they  differ  from  the  pancreatic  ferment  by  acting  best  in  an 
acid  medium.  The  conditions  for  the  solution  of  the  coagulated 
myosin  are  therefore  present,  as  the  reaction  of  the  rigored 
muscles  is  acid. 

*  An  interesting  side-issue  on  the  subject  has  been  taken  up  by  Fletcher 
(Jour,  of  Phys.,  1902,  vol.  xxviii.,  p  474).  He  has  shown  that  an  abundant 
supply  of  oxygen  delays  the  onset  of  both  rigor  mortis  and  fatigue. 

t  Zeit.  f.  physiol.  Chem.,  1901,  vol.  xxxii.,  pp.  341,  531  ;  Jour,  of  Phys., 
1904,  vol.  xxx.,  p.  155. 


LECTURE  II 

HEAT  RIGOR   OF    MUSCLE.      EUGLOBULINS  AND 
PSEUDO-GLOBULINS 

THE  question  must  have  arisen  in  your  minds  while  I  was 
speaking  of  the  muscle  proteids  in  my  last  lecture,  whether 
what  is  found  in  a  saline  extract  of  muscle,  or  in  the  muscle 
juice  pressed  forcibly  from  the  tissue,  really  corresponds  to  what 
is  actually  present  in  the  intact  muscle  itself. 

Such  an  attitude  of  mind  would  have  been  most  commend- 
able, for  it  is  only  by  searching  for  and  meeting  any  objections 
which  can  be  raised,  that  we  can  hope  to  arrive  at  the  truth  in 
any  scientific  problem. 

The  anatomist  is  able  to  see  and  describe  much  of  the 
structures  of  an  animal  organism  without  destroying  its  life. 
The  chemist  is  able  to  explore  the  fields  of  pure  chemistry 
with  his  test-tubes  and  retorts,  and  is  able  with  precision  to 
discover  and  formulate  natural  laws.  But  it  is  when  the 
physiologist  begins  to  investigate  by  chemical  methods  the 
realm  of  living  nature,  that  his  great  difficulties  arise.  It  is 
impossible  for  him  to  say,  for  instance,  in  his  observations  and 
experiments  with  protoplasm,  whether  what  he  finds  is  a  true 
picture  of  the  living  substance,  or  whether  he  is  dealing  with  the 
ruins  of  what  he  has  killed  by  his  reagents.  He  usually  decides 
in  favour  of  the  latter  view.  If  he  uses  strong  and  violent 
reagents,  he  will  assuredly  produce  more  destruction  than  when 
he  employs  gentler  measures,  and  the  employment  of  such  a 
reagent  as  physiological  saline  solution  is  therefore  the  method 
which  secures  the  most  trustworthy  results. 

I  hope,  however,  in  the  case  of  the  muscular  proteids,  to  be 

13 


14  HEAT  RIGOR  OF  MUSCLE  [LECT. 

able  to  convince  you  that  these  are  not  artifacts,  but  really  exist 
in  the  muscle  itself.  The  observations  upon  which  I  rely  to 
prove  this,  are  those  which  were  first  performed  by  Brodie  and 
Richardson*  on  heat  rigor. 

When  a  muscle  is  gradually  heated,  at  a  certain  temperature 
it  is  killed,  and  loses  its  irritability  ;  at  the  same  time  it  contracts 
permanently.  This  phenomenon  is  known  as  heat  rigor,  and  is 
due  to  the  coagulation  of  the  proteid  material  of  the  muscle.  If 
a  tracing  is  taken  of  this  shortening,  it  is  found  that  it  does  not 
take  place  all  at  once,  but  in  a  series  of  steps,  and  the  various 
steps  correspond  to  the  coagulation  temperatures  of  the  various 
proteids,  which  may  be  separated  by  the  process  of  fractional 
heat  coagulation  in  a  saline  extract  of  muscular  tissue.  The 
first  shortening  occurs  at  the  coagulation  temperature  of  para- 
myosinogen  (47°  to  50°  C.),  and  if  the  heating  is  continued,  a 
second  shortening  occurs  at  56°  to  58°  C,  the  coagulation 
temperature  of  myosinogen. 

This  is  very  well  shown  in  the  tracing  (Fig.  i)  taken  from 
Brodie's  paper.  The  first  contraction  began  at  43°,  became 
most  energetic  at  47°,  and  had  finished  at  50°  C.  The  length  of 
the  muscle  then  remained  stationary  until  the  temperature  of 
58°  C.  was  reached,  when  once  more  you  see  shortening  begins. 
This  tracing  illustrates  the  typical  effect  of  heating  a  muscle 
from  a  warm-blooded  animal,  and  those  of  you  who  have  the 
curiosity  to  consult  the  paper  I  have  referred  to  will  find 
numerous  other  similar  tracings  which  bring  out  various  points 
of  detail. 

When,  however,  we  examine  the  tracings  from  the  muscle  of 
a  cold-blooded  animal  like  a  frog,  we  find  that  there  are  three 
instead  of  two  steps  in  the  contraction  ;  they  occur  roughly  at 
40°,  47°,  and  56°  C.  You  will  see  how  exactly  this  corresponds 
with  what  we  have  learnt  from  our  previous  study  of  the  muscle 
plasma  of  the  frog  ;  the  muscle  plasma  of  the  cold-blooded 
animal  contains  an  additional  proteid,  namely,  soluble  myosin. 
and  as  this  is  coagulated  by  heat  at  40°  C.,  it  accounts  for  the 

*  Phil.  Trans,  of  the  Royal  Society,  1899,  13.  vol.  191,  p.  127.  Con- 
firmatory work  in  the  same  direction  has  since  been  published  by  Vernon 
and  others  in  connection  with  other  forms  of  muscular  tissue. 


ii J  HEAT  RIGOR  OF  MUSCLE  15 

first  step  in  the  shortening.  This  is  the  experiment  I  have 
selected  to  show  you,  and  we  are  sufficiently  fortunate  to  have 
Dr  Brodie  here  to  manipulate  his  own  apparatus.  We  have 
thought  it  best  to  select  the  frog,  because  an  experiment  with 
the  muscles  of  a  warm-blooded  animal  is  a  matter  of  some 
difficulty  to  bring  ofT  successfully.  In  warm-blooded  muscles  it 
is  so  difficult  to  get  the  experiment  under  way  before  the 


FlG.  i. — Fresh  gastrocnemius  of  mouse.  Each  break  on  the  curve  represents  a  rise  of 
2°  C.  Total  duration  of  experiment,  40  minutes.  Magnification,  10.  Initial 
length  of  muscle,  13  mm.  Amount  of  the  first  contraction,  1.7;  of  the  second, 
2.4  mm.  (Brodie  and  Richardson.) 

process  of  rigor  mortis  sets  in,  and  this  naturally  upsets  any 
investigation  of  the  myosin-precursors. 

It  is  necessary  to  select  a  very  slender  muscle,  in  order  to 
ensure  that  all  parts  of  it  are  simultaneously  at  the  same  temper- 
ature, and  this  obliges  us  to  have  apparatus  as  light,  free  from 
friction,  and  as  easily  movable  as  possible. 

You  see  now,  the  little  sartorius  in  a  vessel  of  salt  solution, 
over  a  water-bath  the  temperature  of  which  is  going  to  be 
gradually  raised ;  the  muscle  is  fixed  at  its  lower  end ;  the 
upper  end  is  tied  to  a  fine  glass  thread  which  pulls  down  a  straw 
when  the  muscle  shortens.  You  will  be  able  after  the  lecture  to 
examine  the  arrangement  by  which  this  straw  actuates  the 


1 6  HEAT  RIGOR  OF  MUSCLE  [LECT. 

movement  of  a  tiny  mirror  from  which  a  spot  of  light  is 
reflected  on  to  this  screen.  If  we  wished  to  obtain  a  permanent 
record  of  the  contraction,  the  spot  of  light  would  be  made  to  fall 
on  a  slowly  travelling  photographic  plate,  and  this  was  the  way 
in  which  Dr  Brodie  obtained  the  tracings  of  which  I  have  shown 
you  a  sample  (shown  at  the  lecture  as  a  lantern  slide).  The 
breaks  on  what  would  otherwise  have  been  a  continuous  line 
were  produced  by  means  of  a  little  shutter,  which  at  intervals  of 
every  two  degrees  rise  of  temperature  was  closed  for  a  few 
seconds.  These  breaks  render  it  easy  to  read  the  temperature 
on  the  tracing. 

Dr  Brodie's  demonstration,  however,  will  to-day  be  simpler ; 
you  will  not  have  to  wait  until  he  has  developed  a  photographic 
plate,  but  when  the  lights  are  turned  down,  you  will  see  the 
spot  of  light  travelling  up  the  stationary  graduated  screen  as  the 
muscle  shortens.  (Dr  Brodie  then  began  to  heat  the  water-bath, 
and  called  out  its  temperature,  degree  by  degree.)  The 
temperature  of  the  salt  solution  by  which  the  muscle  is  sur- 
rounded was  originally  that  of  the  room  12°  C.  It  has  now 
reached  30°  C.,  and  the  spot  of  light  is  still  stationary  at  the  zero 
mark  of  the  graduation.  The  temperature  is  being  elevated  rather 
more  rapidly  than  is  desirable  in  an  exact  experiment,  but  I  have 
no  doubt  that  this  concession  to  your  patience  will  not  prevent  you 
seeing  the  main  phenomena.  Anyone  who  has  any  experience  in 
determining  the  coagulation  temperatures  of  proteid  solutions,  will 
know  that  a  few  degrees  before  the  heat  is  sufficient  to  cause  the 
appearance  of  visible  flocculi,  there  is  an  opalescent  state  of  the 
fluid  which  gradually  deepens.  You,  in  fact,  saw  this  for  your- 
selves in  my  experiment  on  fractional  heat  coagulation  at  my  first 
lecture.  I  expect  we  shall  see  much  the  same  sort  of  thing  in 
this  experiment ;  although  the  temperature  of  coagulation  of  the 
first  proteid  (soluble  myosin)  is  about  40°  C.,  there  will  be  some 
contraction  corresponding  to  the  stage  of  opalescence  before  we 
reach  that  temperature.  My  expectation  is  now  being  verified  ; 
the  temperature  is  now  35°  C,  and  already  the  spot  of  light  has 
started  slowly  on  its  upward  journey,  but  it  is  nearly  40°  C. 
before  the  contraction  becomes  energetic.  The  temperature  is 
still  rising,  but  the  spot  of  light  is  again  stationary.  Now  once 


li.]  HEAT  RIGOR  OF  MUSCLE  17 

more  it  begins  to  move,  and  the  temperature  is  45°  C.  This 
second  contraction  is  over  by  the  time  we  reach  50°,  and  the 
third  and  final  step  in  the  shortening  takes  place  between  53° 
and  58°  C.  You  may  be  a  little  disappointed  at  the  small 
amount  of  shortening  that  has  occurred  at  the  second  and  third 
as  compared  with  that  in  the  first  step.  This,  however,  is 
merely  due  to  my  desire  to  make  the  experiment  less  tedious. 
The  rapidity  of  the  heating  caused  such  an  energetic  contraction 
when  the  first  proteid  coagulated,  that  the  effect  of  the  second 
and  third  coagulation  is  rendered  comparatively  insignificant  to 
look  at.  If  we  had  allowed  time  for  relaxation  to  occur  after  the 
first  contraction,  or  if  we  had  passively  stretched  the  muscle,  the 
subsequent  contractions  would  have  had  an  opportunity  of 
exhibiting  themselves  in  a  more  prominent  way.  We  did  not 
like  to  risk  the  device  of  stretching  the  muscle,  because  one  is  so 
apt  to  break  such  a  slender  object.  It  gets  very  brittle  after  the 
process  of  heat  rigor  has  set  in.  Nevertheless,  in  spite  of  these 
drawbacks,  you  have  seen  the  main  facts  verified. 

Fig.  2  shows  one  of  Brodie  and  Richardson's  photographic 
records  from  a  frog's  sartorius. 

We  have  hitherto  drawn  a  distinction  in  the  process  of  heat 
coagulation  between  the  formation  of  flocculi  and  the  preliminary 
stage  of  opalescence.  There  is  no  real  distinction  between  the 
two.  One  merges  into  the  other  imperceptibly.  If  one  finds  in 
a  solution  of  a  proteid  that  opalescence  begins  at  a  certain 
temperature,  and  the  separation  of  flocculi  at  a  higher  temperature, 
this  does  not  mean  that  the  phenomenon  at  the  higher  tempera- 
ture is  of  a  different  nature  from  that  which  occurs  at  the  lower ; 
for  if  the  heating  is  continued  long  enough,  flocculi  will  form  at 
the  lower  temperature.  Hewlett  *  puts  the  explanation  of  this 
very  well  as  follows  : — 

u  In  an  aqueous  solution  of  a  proteid  even  when  concentrated, 
there  are  but  comparatively  few  molecules  of  proteid.  The 
temperature  of  the  solution  represents  the  average  energy  of  all 
the  molecules.  When  opalescence  appears,  we  may  suppose 
that  the  changes  which  convert  soluble  into  coagulated  proteid 
have  taken  place  in  some  proteid  molecules,  there  being  a  few 
*  Jour,  of  Phys.,  1892,  vol.  xiii.,  p.  496. 

B 


i8  HEAT  RIGOR  OF  MUSCLE  [LECT. 

which  happen  to  have  attained  greater  velocity  than  that  repre- 
sented by  the  average.  Owing  to  the  irregular  collisions  of  the 
molecules,  their  energies  or  velocities  are  constantly  changing, 
'and  in  time  each  proteid  molecule  will  in  its  turn  attain  the 


V     o 


TlG.  2. —  Frog's  sartorius  which  had  been  heated  to  34°  C.  for  sixty-five  minutes 
twenty-four  hours  previously.  It  was  then  kept  in  cold  dilute  blood,  and  on  the 
following  morning,  although  somewhat  opaque,  it  was  found  to  be  still  irritable. 
It  was  then  passively  stretched,  and  the  above  tracing  taken.  Each  break  in  the 
curve  records  a  rise  of  2°  C.  There  is  a  contraction  commencing  at  34°  C., 

;  ,  although  much  less  than  that  given  by  a  fresh  muscle.  A  second  contraction 
begins  at  47°-48°  C.,  which  is  more  marked  than  in  a  fresh  muscle,  because 
.  ,  of  the  preliminary  stretching.  On  account  of  the  large  amount  of  contraction 
previously  produced,  the  third  and  final  shortening  at  56°-58°  C.  is  not  well- 
marked.  (Brodie  and  Richardson.) 

velocity  necessary  for  coagulation  to  take  place.  The  more 
dilute  the  solution  the  longer  will  this  take,  and  in  any  case  the 
process  must  be  a  slow  one,  on  account  of  the  comparatively 
small  number  of  proteid  molecules.  Raise  the  temperature  a 
degree  or  two,  and  the  number  of  molecules  at  any  given  time 
which  have  attained  the  velocity  necessary  for  coagulation  to 


ii.]  HEAT  RIGOR  OF  MUSCLE  19 

take  place  is  much  increased,  and  coagulation  becomes  more 
rapid.  Now  flocculi  are  merely  aggregations  of  fine  particles, 
these  being  aggregations  of  molecules,  and  if  the  particles  form 
at  a  rapid  rate,  then  flocculi  soon  appear,  as  is  the  case  when  the 
temperature  is  raised  above  that  of  opalescence,  while  if  the 
temperature  is  maintained  at  that  of  opalescence,  particles  form 
only  slowly,  and  it  will  be  a  long  time  before  flocculi  are  seen." 

We  have  noticed  that  in  experiments  like  those  of  Brodie 
on  heat  rigor  there  is  corresponding  evidence  that  coagulation^, 
is  a  gradual,  not  a  sudden,  process,  and  when  we  remember  that 
the  contractile  substance  of  muscle  is  a  semi-fluid  material,  we 
can  at  once  apply  Hewlett's  explanation  to  it  also. 

The  temperature  at  which  the  separation  of  flocculi  occurs 
when  a  solution  of  soluble  myosin  is  heated  at  the  usual 
somewhat  rapid  rate  is  40°  C,  but  Brodie  and  Richardson  found 
that  in  saline  extracts  of  frog's  muscle,  the  soluble  myosin  can 
b^  completely  coagulated  at  34.5°  C.,  provided  the  temperature 
is  maintained  at  that  point  for  a  sufficient  length  of  time. 
In  a  corresponding  way  they  showed  that  in  an  experiment  on 
heat  rigor  the  first  step  in  the  shortening  can  be  entirely 
finished  at  a  temperature  of  35°,  provided  again  sufficient  time  is 
given. 

This  consideration  is  not  merely  of  theoretical  interest ;  it  is 
one  of  immense  practical  importance,  and  I  will  ask  you  to 
bear  it  in  mind  until  we  come  later  on  in  our  study  of 
hyperpyrexia  to  one  of  its  applications. 

There  is  another  point  of  still  greater  interest  arising  out  of 
Brodie's  work  on  heat  rigor,  which  is  this.  What  temperature  is 
necessary  to  destroy  the  irritability  of  a  muscle?  Must  we 
cook  it  so  thoroughly  that  all  its  proteids  are  coagulated  ?  or  is 
it  only  necessary  to  raise  the  temperature  sufficiently  high  to 
coagulate  the  proteid  which  has  the  lowest  temperature  of  heat 
coagulation?  Or  is  the  requisite  temperature  somewhere 
between  the  two  extremes  ?  The  answer  to  this  question  is 
that  the  muscles  lose  their  irritability  after  the  first  step  in  the 
shortening  has  occurred.  We  see,  therefore,  that  when  one  of 
the  muscular  proteids  has  been  coagulated,  the  living  substance 
as  such  is  destroyed.  It  is  therefore  incontrovertible  that 


20  HEAT  RIGOR  OF  MUSCLE  [LECT. 

although  it  is  convenient,  and  from  some  points  of  view 
instructive  and  necessary,  to  regard  the  muscle  proteids  as 
separate  units,  they  are  not  really  independent.  The  unit  is 
protoplasm,  and  if  one  of  its  essential  constituents  is  destroyed, 
protoplasm  as  such  ceases  to  exist. 

You  will  realise  without  waiting  for  me  to  discuss  the 
harmful  influences  of  extremely  high  body  temperatures,  that 
this  is  a  point  for  the  practical  physician  ;  it  is  also  of  interest 
to  the  comparative  physiologist,  and  is  an  evidence  how 
different  kinds  of  organisms  are  fitted  to  their  environment. 
Frogs  are  much  more  easily  killed  by  an  elevated  temperature 
than  warm-blooded  animals,  simply  because  their  living  tissues 
contain  a  proteid  which  coagulates  so  easily  under  the  influence 
of  heat.  If  a  man  were  suddenly  provided  with  frog's  muscles, 
and  his  normal  temperature  of  37°  C.  was  still  maintained,  you  can 
understand  that  he  would  not  live  long.  On  the  other  hand, 
a  bird  has  a  normal  temperature  much  higher  than  that  of  a 
man,  namely  about  42°  C,  which  is  getting  dangerously  near 
to  the  coagulation  temperature  of  para-myosinogen,  but  in  the 
bird  heat  rigor  does  not  occur  until  the  temperature  is  raised  to 
53°  C.  It  is  evident  that  in  the  comparatively  elevated  tempera- 
ture necessary  to  cause  the  first  coagulation  in  birds'  muscle 
plasma  we  have  another  instance  of  biological  adaptation. 

Since  the  foregoing  was  written,  Simin*  has  performed  similar  experi- 
ments on  the  heat  rigor  of  heart  muscle.  In  frog's  heart  muscle,  the  first 
step  in  the  process  occurs  at  43°  C.,  whereas  in  mammals  it  occurs  at  46°  C. 
Alter  rigor  mortis  has  set  in,  the  first  step  in  both  cases  is  abolished. 
Brodie  also  found  the  absence  of  the  first  contraction  the  most  marked 
feature  which  distinguishes  rigored  voluntary  muscle  from  living  muscle. 

Euglobulins  and  Pseudo-Globulins 

For  the  remainder  of  the  hour,  I  will  ask  you  to  consider 
with  me  another  question  which  is  related  to  a  study  of  the 
muscle  proteids.  The  close  connection  between  physiology  and 
pathology  I  have  already  alluded  to,  and,  as  a  rule,  a  knowledge 
of  pathological  processes  is  a  sequel  to  that  of  the  normal. 
But  occasionally  it  is  the  other  way  round,  and  from  what  I 
*  Ceniralb.  f.  Phys.,  1904,  vol.  xviii.,  p.  89. 


ii.]  EUGLOBULINS  AND  PSEUDO-GLOBULINS  21 

have  now  to  say,  you  will  understand  how  the  consideration 
of  a  pathological  problem  led  me  to  take  up  a  physiological 
question. 

A  few  years  ago  I  was  called  upon  to  open  a  discussion  at 
the  Pathological  Society,  London,  on  the  proteids  which  may 
occur  in  the  urine,  and  I  took  occasion  to  point  out  that  the 
presence  of  albumin  in  the  urine  in  greater  quantity  than  the 
globulins  of  the  blood  when  the  damage  to  the  kidney  cells  is 
comparatively  slight  may  possibly  be  a  question  of  molecular 
size.  Experiments  by  Gottwalt*  had  previously  shown  that 
albumin  passes  under  pressure  through  the  membrane  of  a 
dialyser  more  readily  than  globulin,  and  Dr  T.  G.  Brodie  stated 
at  the  discussion  to  which  I  have  just  referred,  that  some 
experiments  he  had  performed  on  perfusion  of  the  isolated 
kidney  supported  the  theory  that  the  molecules  of  the  globulins  x 
are  larger  than  those  of  the  albumins. 

Considering  the  difficulty  of  making  direct  observations  on 
the  molecular  size  of  proteids,  it  is  important  to  utilise  all 
indirect  methods  for  this  purpose.  The  globulins  are,  as  is 
well  known,  more  readily  salted  out  of  their  solutions  than  the 
albumins ;  they  coagulate,  as  a  rule,  at  lower  temperatures,  and 
some  of  them,  like  fibrinogen  and  para-myosinogen,  pass  easily 
into  insoluble  modifications.  All  these  facts  point  to  an  extreme 
colloidal  condition. 

If  now  we  compare  the  colloidal  with  the  crystalline  carbo- 
hydrates, we  see  that  the  colloidal  members  of  the  group  are 
readily  salted  out  from  solution,  whereas  the  crystalline  ones 
are  not.  The  dextriris  which  occupy  an  intermediate  place 
between  the  two  divisions  are  less  readily  salted  out  than  the 
colloidal  carbohydrates,  like  starch  and  glycogen.  The  facility  w 
with  which  the  carbohydrates  are  precipitable  by  alcohol  runs 
parallel  to  this ;  starch  and  glycogen  require  a  comparatively 
small  amount  of  alcohol  to  precipitate  them ;  the  dextrins 
require  more,  while  the  crystalline  carbohydrates  or  sugars  are 
soluble  in  alcohol. 

It  therefore   became   interesting  to   ascertain   whether  the 

proteids  form   a  series   comparable   to    that   described    in    the 

*  Zeit.f.physiol.  Chem.,  vol.  iv. 


22  EUGLOBULINS  AND  PSEUDO-GLOBULltfS        [LECT. 

case  of  the  carbohydrates.  The  difficulty  of  precipitating  the 
proteids  whose  molecules  are  known  to  be  comparatively  small 
(proteoses  and  peptones)  by  means  of  alcohol  is  a  matter  of 
common  experience,  and  some  members  of  this  group  derived 
from  the  vegetable  world  are  even  stated  to  be  soluble  in 
alcohol.  If,  therefore,  it  can  be  shown  that  the  globulins  require 

-a  lower  percentage  of  alcohol  to  precipitate  them  than  the 
albumins,  we  have  an  additional  piece  of  evidence  in  favour  of 
the  view  that  the  globulins  have  larger  molecules  than  the 
albumins.  This  is  the  question  which  I  asked  Miss  Tebb  to 
investigate  in  my  laboratory,  and  it  will  be  seen  that  the  answer 
obtained  was  an  affirmative  one. 

Since  this  work  was  started,  a  paper  appeared  by  Pauli  and 
Rona,*  who  also  pointed  out  the  correspondence  between  the 
precipitation  of  colloids  by  alcohol  and  by  salts,  but  actual 
quantitative  results,  such  as  Miss  Tebb  obtained,  are  not  given. 
The  experiments  were  carried  out  with  four  typical  groups 
of  proteids,  namely,  those  of  blood,  of  egg-white,  of  milk,  and 
of  muscle.  It  is  the  inclusion  of  the  muscular  proteids  in  this 
research  that  has  led  me  to  give  here  a  general  account  of  the 
results  obtained. 

In  the  case  of  the  blood,  the  proteids  present  in    solution 

-  are  :  (i)  fibrinogen,  the  globulin  which  is  the  precursor  of  fibrin  ; 
(2)  serum  globulin ;  and  (3)  serum  albumin.  The  second  and 
third  in  this  list  are  present  in  the  serum,  after  blood-clotting 
has  taken  place.  Many  years  ago,  I  pointed  out  that  the 
u  bstance  called  serum  albumin  of  some  animals  can  be  separated 
by  fractional  heat  coagulation  into  two,  or  in  some  cases  into 
three  proteids  ;  but  as  it  is  difficult  to  find  any  further  difference 
except  that  of  heat  coagulation  temperature  between  them,  the 
question  whether  they  are  really  distinct  proteids  or  not  is  still 
sub  judice ;  and  for  our  present  purpose,  serum  albumin  may 
be  provisionally  regarded  as  a  single  substance. 

More  recent  research  has  shown  that  serum  globulin  is  not 
a  single  proteid.     The  proteid  which  is  salted  out  from  serum 
by  means  ol  half  saturation  with  ammonium  sulphate,  or  com- 
plete  saturation   with    magnesium    sulphate    really  consists   of 
*  Beitriige  chem.  Phys.  u.  Path.,  1902,  vol.  ii.,  p.  i. 


ii.]       '          EUGLOBUL1NS  AND  PSEUDO-GLOBULINS  23 

two  proteids ;  one  of  these  is  insoluble  in  water,  that  is  to  say, 
it  is  precipitable  by  dialysing  away  the  salt  from  its  solutions  : 
it  therefore  fulfils  all  the  definition  of  a  true  globulin,  and  is 
called  euglobulin.  The  other,  known  as  pseudo-globulin,  is  not 
precipitable  by  dialysis. 

In  the  investigation  of  serum  quantitatively,  it  is  now  usual 
to  estimate  the  proteids  in  three  fractions  :  euglobulin,  as  one 
would  expect,  is  most  readily  salted  out,  and  is  brought  down 
by  one-third  saturation  with  ammonium  sulphate ;  in  the 
filtrate,  pseudo-globulin  is  precipitated  by  raising  the  amount  of 
salt  to  the  point  of  half  saturation  ;  the  third  fraction  consists 
of  serum  albumin,  full  saturation  with  the  salt  being  necessary 
to  throw  it  out  of  solution. 

I  need  hardly  assure  you  that  these  refinements  are  not 
invented  by  physiologists  for  the  purpose  of  making  the 
student's  life  more  irksome.  As  knowledge  progresses,  it  often 
becomes  more  complex  for  a  time.  This  is  not  the  occasion  for 
one  to  describe  recent  researches  on  the  blood  in  relation  to  the 
subject  of  immunity  and  similar  questions.  I  will  only  say  that 
the  proteid-like  substances  known  as  antitoxins,  precipitins  and 
the  like  have  been  to  a  certain  extent  separated  out  by 
fractional  precipitation  with  salts,  for  they  tack  themselves  on 
to  the  various  proteid  fractions  in  different  ways.  This  line  of 
research,  still  in  its  infancy,  holds  out  great  promise  for  the 
future. 

Miss  Tebb  soon  found  as  her  work  progressed  that  the 
globulin  of  white  of  egg  must  also  be  divided  into  an  eu-  arid  a 
pseudo-  fraction. 

With  this  introduction  we  may  now  pass  to  her  results. 
The  following  numbers  represent  the  amount  of  alcohol  per 
cent  necessary  to  produce  complete  precipitation  of  the  various" 
proteids. 

Blood  Proteids — 

Fibrinogen  •.>   '        "'*,  .  ':.   ;  '.:.':  '   .    :      30 

Serum  euglobulin      .             .  '  ...  .  ;'      25 

Serum  pseudo-globulin         .  ••:..  '    .         50 

Serum  albumin          .:            .  "           .  50 


44  &UGLOBVLINS  AND  PSEUDO-GLOBULINS        [LECT 

Egg-  White  Proteids — 

Egg  euglobulin          .  .             .  -^  20 

Egg  pseudo-globulin  .  I       '-„'..          .    .  65 

Egg  albumin              .  .,          ...'..-..          .  40 

Muscle  Proteids — 

Para-myosinogen       «  .  .  .20 

Myosinogen  .  ,  ..  80 

Milk  Proteids — 

Caseinogen — The    main  bulk   is    brought    down  by  45  per 

cent,  of  alcohol,  but  it  is  not  completely  precipitated  by 

90  per  cent. 
Lact-albumin — The  main  bulk  is  brought  down  by  65,  and 

the  last  traces  by  80-85  Per  cent,  of  alcohol. 

We  therefore  see  from  these  results  : — 

1.  That  the  true  globulins  of  blood  and  egg-white  require 
considerably   less    alcohol    to    precipitate    them    than    do    the 
albumins. 

2.  Although  the  pseudo-globulins  are  more  readily  salted 
out  from  their  solutions  than  are  the  albumins,  and  less  readily 
than  the  euglobulins,  the  precipitability  by  alcohol  does  not  run 
quite    parallel    to    this.     On    the   whole    the    pseudo-globulins 
resemble  the  albumins  in  their  precipitability  by  alcohol,  but  in 
one  case,  that  of  egg-white,  the  albumin   is  more  readily  pre- 
cipitable  by  alcohol  than  is  the  pseudo-globulin. 

The  results  obtained  with  the  proteids  of  milk  were  a  little 
unexpected :  lact-albumin  is  precipitable  with  difficulty  by 
alcohol,  and  so  falls  into  line  with  the  other  albumins ;  but 
caseinogen,  which  one  would  rather  have  anticipated  to  behave 
like  a  globulin,  requires  also  a  considerable  amount  of  alcohol 
to  precipitate  it  entirely ;  most,  however,  is  thrown  out  of 
solution  by  a  comparatively  small  amount  of  alcohol. 

The  results  with  the  muscle  proteids  are  those  which  will 
interest  us  most.  Both  (and  especially  para-myosinogen)  are 
readily  salted  out  from  their  solutions,  but,  as  we  have  already 
seen,  myosinogen  is  soluble  in  water,  and  we  have  described  it 
as  an  atypical  globulin.  It  is  not  stretching  the  use  of  terms  too 


II.]  EUGLOBULINS  ANb  PSEUDO-GLOBULINS  2$ 

much  to  say  that  para-myosinogen  is  the  euglobulin  of  muscle  ; 
like  the  other  euglobulins,  it  is  readily  precipitable  by  alcohol ; 
myosinogen,  the  pseudo-globulin  of  muscle,  requires  much  more 
alcohol  to  precipitate  it  entirely. 

The  main  conclusion  drawn  from  this  work  is,  that  there  is 
distinct  though  indirect  evidence  that  the  true  globulins  have 
larger  molecules  than  the  pseudo-globulins  and  albumins. 

Among  some  subsidiary  points  worked  out  by  Miss  Tebb,  the  following  is 
of  importance  : — The  prolonged  action  of  alcohol  renders  proteids  insoluble. 
The  euglobulins  are  most  readily  rendered  insoluble  in  this  way  ;  the  pseudo- 
globulins  and  caseinogen  come  next,  whilst  of  the  proteids  investigated  the 
albumins  are  the  most  difficult  to  convert  into  insoluble  modifications  by 
alcohol.  It  was  previously  well  known  that  proteids  of  still  smaller  mole- 
cular size  (proteoses  and  peptone)  not  only  require  a  large  amount  of  alcohol 
to  precipitate  them  entirely,  but  also  are  not  rendered  insoluble  by  prolonged 
contact  with  that  reagent. 

Considering  the  way  in  which  this  investigation  originated  from  a  study 
of  proteids  in  pathological  urine,  it  is  interesting  in  conclusion  to  note  some 
results  published  last  year  by  J.  Joachim.*  He  finds  that  when  serum 
globulin  occurs  in  urine,  it  is  almost  exclusively  pseudo-globulin  which  is 
present. 

*  Pfliiger's  Archiv,  1903,  vol.  xciii.,  p.  558. 


LECTURE  III 

•  ".:  ''  -  . '  -    .   i     -.<  •• 

THE   PIGMENTS   OF   MUSCLE.      PROPERTIES   OF    NUCLEO- 
PROTEIDS.      THE   FERMENTS   OF   MUSCLE 

THE  voluntary  muscles  of  a  mammal  are  usually  divisible  into 
two  main  varieties,  which  are  respectively  called  the  pale  and 
the  red  muscles.  These  are  readily  distinguishable  to  the  naked 
eye  by  their  colour,  to  the  microscope  by  certain  points  of  histo- 
logical  difference,  and  in  physiological  experimentation  it  is 
found  that  the  contraction  period  is  longer  in  the  red  than  in  the 
pale  muscles. 

The  rabbit  is  generally  used  as  the  animal  in  which  to  show 
this  in  demonstrations,  because  in  this  animal  the  distinction  is 
so  well  seen  ;  the  majority  of  the  muscles  are  of  the  pale  variety, 
but  in  a  few  muscles  the  fibres  are  almost  exclusively  of  the  red 
variety.  I  have  here  the  leg  of  a  rabbit  from  which  all  the  blood 
has  been  washed  out  by  a  stream  of  salt  solution.  We  have 
therefore  excluded  any  coloration  due  to  the  blood.  If  you 
look  at  it,  you  readily  see  how  such  muscles  as  the  semi- 
membranous  and  crureus  stand  out  red  on  the  pale  background 
of  the  surrounding  flesh. 

I  have  taken  a  small  piece  of  one  of  these  red  muscles,  and 
after  mincing  it,  ground  it  up  with  some  salt  solution ;  the 
filtered  extract  I  have  placed  in  front  of  a  spectroscope,  and 
after  the  lecture  you  will  have  the  opportunity  of  identifying  the 
two  well-known  absorption  bands  of  oxyhaemoglobin. 

There  is  no  doubt  that  the  coloration  is  due  to  blood- 
pigment  actually  contained  within  the  muscle  plasma. 

There   is    some   colour   in   the  so-called  pale  muscles,  and 

26 


LECT.  in.]  THE  PIGMENTS  OF  MUSCLE  2? 

probably  here  we  have  to  deal  with  the  same  pigment  in  smaller 
quantities. 

Some  years  ago,  however,  Dr  MacMunn*  stated  that  the 
main  pigment  of  muscles  is  a  special  colouring  matter,  to  which 
he  gave  the  name  myohcematin.  He  found  it  also  in  many 
invertebrate  animals,  in  the  blood  of  which  no  haemoglobin  is 
contained.  He  found  also  in  other  tissues  somewhat  similar 
pigments,  and  the  name  histo-hcematins  which  he  gave  to  the 
whole  group  indicates  their  similarity  to  haemoglobin ;  he 
further  considered  that  they  act  as  respiratory  pigments,  and 
showed  in  support  of  this  view  that  their  absorption  bands 
undergo  changes  on  oxygenation  and  reduction. 

Unfortunately,  these  pigments  were  never  separated  out  from 
the  tissues  in  a  pure  condition  and  analysed.  They  were  in  the 
main  detected  and  identified  by  the  spectroscope,  usually  after 
the  tissues  had  been  rendered  transparent  by  the  action  of  such 
reagents  as  glycerin. 

I  will  next  demonstrate  to  you  the  method  by  which  myo- 
haematin  can  be  rendered  visible.  I  have  here  a  piece  of  the 
pectoral  muscle  of  a  pigeon,  a  very  deeply  pigmented  muscle. 
It  has  been  in  glycerin  since  yesterday,  and  is  now  quite  trans- 
lucent ;  I  place  a  thin  layer  of  it  between  two  glass  slides  and 
put  it  in  front  of  the  spectroscope.  After  the  lecture  you  will 
be  able  to  see  its  absorption  bands,  which  are  shown  in  the 
accompanying  diagram  (Fig.  3,  spectrum  i). 

MacMunn  proved  that  the  bands  fade  on  the  application  of 
a  reducing  agent. 

He  introduced  also  another  method  by  which  the  pigment 
may  be  obtained  in  solution,  which  I  will  proceed  to  show  you. 
Again  I  have  taken  the  pigeon's  breast  muscle,  chopped  it  into 
small  pieces,  and  placed  it  in  a  flask  with  excess  of  ether  since 
yesterday.  You  will  see  that  the  ether  is  of  a  distinctly  yellow 
colour ;  but  the  yellow  pigment  is  not  myohaematin,  but  is 
simply  a  fatty  pigment  or  lipochrome.  These  lipochromes  used 
at-  one  time  to  be  called  luteins,  because  the  best  known  member 
of  the  group  was  obtained  from  the  corpus  luteum  of  the  ovary. 
Adipose  tissue,  milk,  blood  serum,  etc.,  yield  similar  pigments 
*  Phil.  Trans.,  1886.  Jour,  of  Phys.,  1887,  vol.  viii.,  p.  51. 


2&  THE  PIGMENTS  OF  MUSCLE  [LECT. 

to  solvents  of  fat.  The  lipochrome  now  dissolved  in  the  ether 
doubtless  comes  from  the  adipose  tissue  and  blood  serum  con- 
tained within  the  muscle. 

I  pour  off  the  yellow  ethereal  fluid,  and  now,  when  I  am 
reaching  the  bottom,  another  fluid  on  which  the  ether  has  been 
floating  comes  away.  The  colour  of  this  aqueous  fluid  is 
reddish;  it  is  rather  cloudy,  for  it  contains  in  suspension  a 
number  of  particles  of  minced  muscle.  I  proceed  now  to 
separate  the  two  fluids  with  a  separating  funnel,  and  we  will 
examine  the  red  aqueous  fluid,  for  it  is  this  that  contains  myo- 
haematin. In  contact  with  ether,  osmotic  processes  have  occurred 
in  the  muscle,  resulting  in  the  passing  out  of  it  of  this  watery 
fluid,  which  carries  some  of  the  pigment  with  it.  It  also  con- 
tains a  certain  amount  of  proteid  and  extractives,  but  those 
need  not  concern  us.  After  filtration,  the  fluid  is  seen  to  be 
perfectly  clear.  I  place  some  of  it  in  front  of  another  spectro- 
scope, and  those  of  you  who  take  the  trouble  of  examining  it, 
will  see  the  absorption  bands  figured  in  spectrum  2  of  Fig.  3.  If 
you  are  familiar  with  the  spectroscopic  appearance  of  haemo- 
globin derivatives,  you  will  observe  that  the  two  bands  seen  are 
something  like  those  of  haemochromogen,  but  are  nearer  the 
violet  end  of  the  spectrum.  This  substance  further  differs  in 
spectroscopic  characters  from  the  myohaematin,  as  seen  by  the 
glycerin  method,  and  so  was  termed  "  modified  myohaematin  "  by 
MacMunn.  A  similar  modification  is  produced  by  subjecting  the 
muscle  to  artificial  gastric  digestion. 

The  question  before  us  is,  whether  myohaematin  is  modified 
haemoglobin.  Levy,*  who  worked  under  Hoppe-Seyler's  direc- 
tion, gave  it  as  his  opinion  that  myohaematin  is  simply  haemo- 
chromogen.  It  is  difficult  to  understand  how  he  can  have 
reached  this  conclusion,  for,  seeing  that  spectroscopic  analysis 
was  the  only  method  employed,  it  is  remarkable  that  he  did 
not  pay  attention  to  the  marked  difference  in  the  position  of  the 
absorption  bands  in  the  two  cases.  The  difference  is  so  evident 
that  you  will,  even  with  the  small  direct  vision  spectroscopes  on 
the  table,  have  no  difficulty  in  seeing  it.  I  have  placed  some 

*  Zeit.  f.  physiol.   Chem.,  vol.   xiii      MacMunn's  reply  is  in  the  same 
volume. 


FlG.  3. — I.  Absorption  spectrum  of  myohaematin  in  muscle  rendered  transparent  by 
glycerin.  2.  Absorption  spectrum  of  modified  myohsematin  obtained  by  ether 
method. 


[To  face  page  28. 


in.]  THE  PIGMENTS  OF  MUSCLE  29 

haemochromogen    prepared    from    blood    in    front    of   another 
spectroscope  to  enable  you  to  compare  the  two. 

Morner  *  has  suggested  that  the  proteid  constituent  of  the 
pigment  may  be  different  in  the  two  cases,  but  this  is  only  a 
hypothesis.  The  subject  is  one  which  would  repay  further 
research.  I  think  myself  that  there  can  be  no  doubt  that  myo- 
haematin  is  a  derivative  of  haemoglobin,  but  whether  the  mus- ' 
cular  tissue  is  capable  of  producing  the  change  in  spite  of  the 
reagents  added,  or  whether  the  reagents  added  are  mainly 
responsible  for  the  change,  one  cannot  at  present  say.  I  am 
led  to  this  conclusion  by  the  following  consideration.  The 
muscles  in  which  I  have  been  able  to  show  you  the  pigment 
were  not  freed  from  blood  before  being  placed  in  glycerin  or 
ether  respectively  ;  and  they  contained  plenty  of  blood  at  the 
start.  Yet,  the  spectrum  of  the  blood  pigment  has  entirely  dis- 
appeared, and  its  place  taken  by  that  of  myohaematin. 

I  am  strengthened  in  this  conviction  by  some  experiments  of 
Copeman.f  He  mixed  defibrinated  and  slightly  diluted  blood 
with  minced  muscle,  and  kept  the  mixture  at  36°  C.  for  nearly 
three  weeks,  contact  with  air  being  prevented.  At  the  end  of 
that  time,  the  spectrum  was  not  distinguishable  from  that 
figured  by  MacMunn  for  myohaematin.  On  heating  to  near 
boiling-point  the  bands  disappear ;  and  this,  I  had  previously 
pointed  out,  was  a  distinctive  feature  of  modified  myohaematin. 
Similar  experiments  carried  out  with  small  quantities  of  liver 
and  other  tissues  macerated  in  blood  resulted  in  the  formation 
of  the  haemoglobin  derivative  nearest,  spectroscopically,  to 
myohaematin,  namely,  haemochromogen. 

Further,    MacMunn   himself   has    shown    that   haemoglobin 
derivatives  like    acid    haematin    and    haematoporphyrin   can  be^ 
obtained  from  myohaematin. 

Properties  of  Nucleo-Proteids 

The  mention  of  nucleo-proteids  among  the  constituents  of 
muscle  leads  me  next  to  speak  in  general  terms  of  these  impor- 

*  Nord.  med.  Ark.,  Stockholm,  Festband,  1897. 

t  Proc.  Phys.  Soc.,  Nov.  8,  1890.    Jour,  of  Phys.,  vol.  xi.,  p.  xxii. 


30  THE  PIGMENTS  OF  MUSCLE  [I.ECT. 

tant  substances.  They  are  not  very  important  in  muscle  from 
the  quantitative  point  of  view.  But  as  they  are  also  found  in 
nervous  structures,  I  want  briefly  to  indicate  some  of  their 
principal  characters  before  we  finally  leave  the  subject  of  the 
muscular  proteids. 

I  then  described  these  substances,  dealing  first  with  their  chemical  char- 
acters and  relationships,  and  the  purine  and  other  substances  obtained  ir 
the  decomposition  of  true  nuclein  in  contradistinction  to  that  of  pseudo-nuclein 
I  then  proceeded  to  deal  with  the  physiological  importance  of  nucleo-proteids, 
and  dwelt  particularly  on  the  way  in  which,  when  injected  into  the  blood 
'stream,  they  cause  intra vascular  clotting.  This  led  me  finally  to  a  demon- 
stration. I  prepared  some  nucleo-proteid  from  a  freshly  removed  kidney 
before  the  class,  by  my  sodium-chloride  method,  dissolved  it  in  dilute 
sodium  carbonate  solution,  and  injected  it  into  the  blood  stream  of  an 
anaesthetised  rabbit.  After  the  death  of  the  animal,  which  occurred  after  the 
injection  of  a  few  cubic  centimetres,  the  intravascular  coagulation  was  found 
to  be  very  complete,  especially  in  the  venous  system.  This  necessarily  led 
me  to  speak  on  the  subject  of  blood  coagulation  in  general.  These  subjects, 
although  appropriate  enough  in  a  spoken  lecture  and  demonstration,  were 
introduced  by  way  of  parenthesis  ;  but  it  is  neither  suitable  nor  necessary  to 
enter  into  these  side  questions  in  a  book  which  professes  to  deal  especially 
with  the  chemistry  of  muscle  and  nerve. 

The  Ferments  of  Muscle 

Ferments  are  substances  which  have  to  a  great  extent 
eluded  the  grasp  of  the  chemist.  All  he  can  say  is,  that  they 
are  probably  proteid-like  in  nature,  and  in  some  cases  the 
proteid  material  with  which  they  are  either  identical  or  united, 
-  is,  as  in  the  case  of  the  fibrin  ferment,  of  the  nucleo-proteid 
variety. 

I  have  already  alluded  to  the  possible  existence  of  a  myosin 
ferment  concerned  in  muscle  coagulation.  I  will  only  add  that 
if  it  does  exist,  it  is  not  identical  with  fibrin  ferment.  Fibrin 
ferment  does  not  hasten  the  clotting  of  muscle  plasma,  nor  does 
myosin  ferment  hasten  the  coagulation  of  blood  plasma.  The 
addition  of  pieces  of  fresh  muscle  and  fresh  tissues  generally 
does  accelerate  the  clotting  of  blood,  or  of  blood  plasma,  and 
this  is,  according  to  some  observers,  due  to  adherent  lymph ; 
this  view  is  disputed  by  Delezenne,  and  the  actual  substance  to 
which  the  result  is  due  must  be  considered  unknown  at  present. 


in.]  THE  FERMENTS  OF  MUSCLE  31 

In  addition  to  this  ferment,  let  me  also  remind  you  of  the 
existence  of  a  proteolytic  enzyme  which,  I  have  already  stated, 
is  probably  concerned  in  the  disappearance  of  rigor  mortis 

(p.    12). 

In  addition  to  these,  there  are  two  others :  (i)  an  amylolytic 
ferment  which  converts  starch  into  sugar  ;  here  again  muscle  is 
not  peculiar,  but  a  similar  enzyme  is  obtainable  in  extracts  of 
most  tissues.  The  final  sugar  formed  is  dextrose  ;  hence  it  is  also 
necessary  to  assume  the  existence  of  another  ferment  (maltase), 
which  converts  maltose  into  dextrose ;  (2)  a  glycolytic,  or  sugar- 
destroying  ferment,  which  is  probably  of  importance  in  carbohy- 
drate katabolism,  and  to  which  Brunton  and  Rhodes*  were  the 
first  to  draw  attention.  Glycolysis  occurs  in  many  tissues,  and 
the  agent  or  ferment  to  which  this  is  due,  is  believed  by  Cohn- 
heimf  to  be  rendered  active  by  the  internal  secretion  of  the* 
pancreas. 

*  Proc.  Roy.  Soc.,  1901,  vol.  Ixviii.,  p.  323. 

t  Zeit.f.physiol.  Chem.,  1903,  vol.  xxxix.,  p.  336  See  also  J.  Feinschmidt 
(Beitriige  chem.  Phys.  Path.,  1903,  vol.  iv.,  p.  511),  on  the  Sugar-destroying 
Ferment  in  Organs. 


LECTURE    IV 


THE   EXTRACTIVES   AND   SALTS   OF   MUSCLE 

THE  extractives  of  muscle  form  a  heterogeneous  group  of 
organic  substances,  which  occur  in  small  quantities,  and  which 
may  be  extracted  by  the  usual  reagents  employed  for  that  pur- 
pose, such  as  alcohol,  ether,  or  water. 

I  will  begin  by  giving  you  a  list  of  the  extractives,  putting 
them  in  two  groups,  non-nitrogenous  and  nitrogenous. 


A.  Non-nitrogenous  extractives. 

1.  Glycogen. 

2.  Dextrin  and  sugars. 

3.  Lactic  acids. 

B.  Nitrogenous  extractives. 

1.  Creatine. 

2.  Creatinine. 

3.  Xanthine. 

4.  Hypoxanthine. 

5.  Uric  acid. 


4.  Inosite. 

5.  Fat. 


6.  Urea. 

7.  Carnine. 

8.  Carnic  acid. 

9.  Inosinic  acid. 
10.  Taurine. 


Nearly  all  of  these  possess  some  special  point  of  interest. 
Many  of  them,  like  glycogen,  sugar,  creatine,  and  urea  open  up 
important  general  questions  of  metabolism.  Some,  like  uric 
acid  and  its  near  chemical  relatives,  would  lead  us  into  patho- 
logical bypaths  if  we  followed  them  up  in  detail.  To  treat 
all  of  these  questions  in  full  would  lead  us  too  far  from  the 


LECT.  iv.]         THE  CARBOHYDRATES  OF  MUSCLE  33 

main  study  in  which  we  are  engaged,  and  exigencies  of  time 
further  compel  me  to  do  little  more  than  indicate  the  main 
characters  of  each  member  of  our  long  list;  and  to  refer  you  to 
the  side  questions  which  they  suggest. 


The  Carbohydrates  of  Muscle 

The  principal  carbohydrate  of  muscular  tissue  is  glycogen. 
It  may  be  extracted  from  muscle  by  boiling-water  (Briicke),  by 
dilute  potash  (Kiilz),  or  by  strong  potash  (Pavy,  Pfliiger).  The 
last-named  method  is  the  one  now  most  generally  employed, 
and  Pfliiger  has  fully  confirmed  Pavy's  statement  that  the 
extraction  by  the  use  of  this  reagent  is  not  only  very  thorough, 
but  there  is  little  or  no  loss  of  glycogen  during  the  process. 
In  a  number  of  somewhat  lengthy  papers  in  recent  volumes  of 
Pfliiger's  Archiv,  it  has  been  shown  that  the  estimations 
previously  made  by  the  older  methods  require  revision,  and 
that  glycogen  is  a  constituent  of  many  tissues  and  organs  where 
its  presence  was  previously  unsuspected.  The  addition  of  a 
comparatively  small  amount  of  alcohol  to  the  extract  precipitates 
the  glycogen,  and  so  it  may  be  separated  from  the  proteid 
matter  in  the  extract,  which  requires  more  alcohol  to  precipitate 
it. 

Glycogen  is  not  equally  distributed  throughout  the  muscu- 
lature, and  estimations  have  been  made  in  various  skeletal 
muscles  and  involuntary  muscles  by  Cramer,  Boruttau, 
Chittenden,  Bizio  and  others.  The  numbers  obtained  possess 
but  little  general  interest.  It  is  more  important  to  study  the 
varying  amount  under  different  circumstances. 

1.  In  starvation. — The  muscle  glycogen   disappears  during 
inanition,  but  much  more  slowly  than  the  hepatic  glycogen.* 

2.  During  work. — The  glycogen  of  the  muscles  disappears 
during   work,   being   probably   transformed    into   sugar   which 

*  Weiss,  Sitzungsber  d.  k.  Akad.  d.  Wissensch.,  Wien,  vol.  64  ;  Aldehoff, 
Zeit.f.  BioL,  vol.  xxv.,  p.  137  ;  Luchsinger  (Dissertation,  Zurich,  1875)  stated 
the  heart  glycogen  disappears  more  slowly  still,  but  Aldehoff  could  not 
confirm  this, 

C 


34 


THE  EXTRACTIVES  AND  SALTS  OF  MUSCLE     [LECT. 


then    undergoes   combustion.     The    following   table  *    may    be 
taken  as  a  type  of  the  results  obtained. 


Percentage  of  Glycogen. 

Percentage  loss  of  Glycogen 
in  Tetanised  Limb. 

In  limb  at  rest. 

In  the  opposite  limb,  made 
to  contract  for  25  to  65 
minutes. 

I 

2 

3 

0.1277 
0.2287 
0.2267 

0.114 
0.1942 
0.1917 

12.76 
15.09 
1544 

This  question  is  one  of  great  importance,  and  opens  up  the 
general  question  of  the  source  of  muscular  energy.  There  are 
some  physiologists  who  still  hold  with  Liebig  that  the  proteids 
are  the  main  source  of  muscular  energy,  whereas  the  exactly 
opposite  view  is  held  by  others,  namely,  that  the  non-nitrogenous 
constituents  are  those  which  are  chiefly  used  up.  The  large 
and  immediate  increase  in  the  discharge  of  carbon  dioxide 
which  occurs  when  a  muscle  contracts,  is  certainly  in  striking 
contrast  with  the  unimportant  increase  in  the  products  of  proteid 
katabolism  which  takes  place,  as  was  first  shown  by  the  classical 
experiment  of  Fick  and  Wislicenus  in  their  historical  ascent  of 
the  Faulhorn  in  1865. 

Those  of  you  who  are  athletes  should  be  specially  interested 
in  this  problem.  As  a  rule,  an  athlete  does  not  trouble  much 
about  the  scientific  reasons  for  his  method  of  training ;  he  is 
guided  in  his  diet  by  what  others  have  found  to  be  successful  in 
the  past,  and  a  good  deal  of  tradition  gets  mixed  with  the  ideas 
that  prevail  on  the  subject.  Generally,  a  large  amount  of  proteid 
food  is  taken,  and  carbohydrates  and  fats  are  knocked  down. 
Experience  shows  that  muscle  works  most  economically  when 
fed  chiefly  on  proteid ;  at  any  rate,  the  waste  in  heat  production 
that  accompanies  the  combustion  of  fat  and  carbohydrate  is  not 
so  great.  We  must,  however,  remember  that  during  recent 
years,  feats  of  great  endurance  have  been  performed  by  men 
who  have  used  mainly  carbohydrates  during  training.  On  most 

*  Manche,  Zeit.  f.  BioL,  vol.  xxv.,  p    163. 


iv.]  THE  CARBOHYDRATES  OF  MUSCLE  35 

subjects  of  physiology,  as  in  politics,  where  there  are  divergencies 
of  opinion,  there  is  generally  truth  on  both  sides.  I  do  not 
intend  to  weary  you  with  the  details  of  this  particular  contro- 
versy, but  will  merely  say  that  the  majority  of  level-headed 
physiologists  are  now  agreed  that  muscular  energy  comes  from 
all  three  classes  of  food  stuffs.  So  long  as  an  ordinary  mixed 
diet  is  taken,  the  diet  which  centuries  of  experience  have  shown 
to  be  most  suitable  for  man,  there  will  be  sufficient  carbonaceous 
food  present  in  the  muscle,  and  the  muscle  chiefly  uses  this 
for  the  energy  required  in  its  contraction.  But  if  this  is  not 
given,  or  the  accumulated  store  of  fat  and  carbohydrate  in  the 
muscle  is  used  up,  then  muscular  work  must  be  maintained  by- 
the  disintegration  of  its  proteid.* 

3.  Paralysis  of  a   muscle,   produced    either   by   cutting   its 
nerve  f  or  its  tendon ,J  causes  an  accumulation  of  glycogen  in  it. 
This  is  the  opposite  to  the  effect  of  work,  and  so  would  have 
been  expected.     Ligature  of  the   arteries   to   a   muscle   leads, 
on  the  other  hand,  to  a  decrease  in  the  glycogen,  especially  if 
oedema  follows  the  operation,  the  accumulated  lymph  leading 
to  saccharification  (Manche  ;  Chandelon). 

4.  Removal  of  the  liver. — Though  some  observers  §  consider 
that  the  muscles  have  a  glycogenic  function  independent  of  that 
of  the  liver,  it  appears  to  be  an  undoubted  fact  that  extirpation 
of  the  liver  leads  to  a  rapid  disappearance  of  the  muscular 
glycogen  (Minkowski).|| 

The  sugar  in  muscle  is  during  life  at  a  minimum  ;  as  it  is 
formed  from  the  glycogen,  it  is  apparently  soon  burnt  up.  After 
death,  as  in  the  liver,  the  amount  of  sugar  increases  as  the 

*  On  the  beneficial  effect  of  feeding  voluntary  muscle  on  sugar,  see  Lee 
(Centralbl.  f.  Phys.,  1901,  vol.  xv.,  p.  482).  Corresponding  effects  on  the 
isolated  mammalian  heart  are  described  by  Locke  (Proc.  Phys.  Soc.,  1904, 
p.  xii.  ;  Jour,  of  Phys.,  vol.  xxxi.).  The  important  sugar  is  dextrose. 

f  Chandelon,  Pfliiger's  Archiv,  vol.  xiii.,  p.  626.  See  also  Manche, 
loc.  cit. 

\  E.  Krauss,  Virchow's  Archiv,  vol.  cxiii.,  p.  315. 

§  Prausnitz,  Zeit.  f.  BioL,  vol.  xxvi.,  p.  377.  Schmelz,  ibid.,  vol.  xxv., 
p.  1 80. 

||  Arch.  f.  exper.  Path.  Pharm.,  vol.  xxiii.,  p.  139.  See  also  Schmelz, 
loc.  cit.;  and  Laves,  Inaug.  Dissertation,  Konigsberg,  1886. 


36  THE  EXTRACTIVES  AND  SALTS  OF  MUSCLE     [LECT. 

glycogen  disappears.  Nasse*  considered  that  the  sugar  is 
maltose  ;  but  the  work  of  Panormoff'f'  with  the  phenylhydrazine 
reaction  showed  it  to  be  dextrose.  That  dextrose  should  be 
found  is  not  to  be  wondered  at,  for  in  my  enumeration  of  the 
ferments  in  muscle  you  will  remember  I  drew  your  attention  to 
an  amylolytic  enzyme,  and  a  maltase  as  well.  Some  few  years 
ago,  Miss  Tebb,  in  my  laboratory,  worked  out  the  properties  of 
the  dextrins  which  are  formed  as  intermediate  products  in  the 
conversion  of  glycogen  into  sugar,  and  showed  that  on  the 
whole  they  are  analogous  to  those  found  during  the  hydrolysis 
of  starch.  We  should  therefore  expect  in  muscle  to  find  not 
only  the  final  product  dextrose,  but  also  the  intermediate  stages 
of  dextrin  and  maltose.  The  careful  work  of  Osborne  and 
ZobelJ  has  in  my  opinion  placed  it  beyond  doubt  that  this 
is  really  the  case.  Although  it  is  therefore  correct  to  use  the 
word  sugars  in  the  plural  in  reference  to  muscle,  we  ought  not 
to  include  inosite  among  them.  Inosite  was  formerly  called 
muscle-sugar,  and  is  present.  But,  as  you  all  know  from  your 
text-books,  inosite,  though  isomeric  with  the  glucoses,  is  really 
a  member  of  the  aromatic  series. 

The  Fat  of  Muscle 

Fat  is  always  obtainable  from  muscular  tissue,  though 
whether  it  occurs  in  the  true  muscular  substance  apart  from  the 
entangled  adipose  tissue,  it  is  difficult  to  say.  Dormeyer  § 
finds  that  after  muscle  has  been  subjected  to  preliminary  gastric 
digestion,  ether  extracts  8.5  per  cent,  more  of  the  total  fat 
obtainable,  and  has  gone  so  far  as  to  say  that  without  such 
preliminary  digestion,  extraction  is  useless  for  quantitative 
purposes.  This  view  has  not  been  entirely  confirmed  by 
subsequent  workers ;  if  the  subdivision  of  the  muscle  is  great 
enough,  and  the  extraction  carried  out  sufficiently  long,  with 
suitable  agitation  during  the  process,  ether  has  been  found 

*  Zur.  Anat.  u.  Phys.  der  quergestreiften  Muskel,  Leipzig,  1882. 
t  Zeit.  f.  physiol.  Chem.,  vol.  xvii. 
%  Jour,  of  Phys.,  1903,  vol.  xxix.,  p.  I. 
§  Pfltiger's  Archiv,  1896,  vol.  Ixv.,  p.  90. 


iv.]  THE  LACTIC  ACIDS  37 

sufficient.  There  is,  however,  no  doubt  that  gastric  digestion  is 
one  means  of  accomplishing^sufficient  subdivision.  Bogdanow* 
believes  that  the  fat  thus  soluble  in  ether  with  difficulty  is  a  real 
constituent  of  the  muscle  plasma,  and  states  that  it  is  richer  in 
volatile  fatty  acids  than  that  from  the  surrounding  connective 
tissue. 

The  only  other  point  of  importance  in  reference  to  this 
question  is  the  statement  by  Leathes,-|-  that  the  red  muscles  of 
the  rabbit  are  richer  in  fat  than  the  pale  muscles  of  the  same 
animal,  or  the  mixed  muscles  of  the  cat. 

The  Lactic  Acids 

Among  the  oxypropionic  acids  with  the  empirical  formula 
C3H6O3,  several  are  at  present  known  to  chemists. 

One  of  these,  called  ethylene  lactic  acid,  has  the  formula 

CH2(OH).CH2.COOH. 

This  is  not  found  in  the  body.J: 

The  remaining  lactic  acids  are  stereo-chemical  isomerides  of 
ethylidene  lactic  acid.  Their  formula  is 

CH3.(CH.OH)COOH. 

They  are  three  in  number,  and  the  differences  between  them 
are  due,  according  to  the  theory  of  le  Bel  and  Van't  Hoff,  and 
as  the  expression  stereo-chemical  implies,  to  the  space  relation- 
ships of  the  atoms. 

They  differ  in  optical  activity,  and  in  the  solubility,  optical 
activity,  and  amount  of  crystallisation  water  in  their  zinc, 
calcium,  lithium,  and  barium  salts. § 

The  three  isomerides  are — 

(a)  The    optically    inactive    acid.     This     is    the     ordinary 

•*  Pfliiger's  Archiv,  vol.  Ixv.,  p.  81. 

t  Proc.  Phys.  Soc.,  1904,  p.  ii.  ;  Jour,  of  Phys.,  vol.  xxxi. 

I  Small  quantities  of  it  were  described  in  muscle  extracts  by  Wislicenus 
(Ann.  d.  Chem.,  1873,  vol.  clxvii.,  p.  302)  ;  but  this  is  not  so  ;  the  acid 
mistaken  for  it  was  acetyl  lactic  acid,  H3CH(C2H3O2)COOH  (Siegfried, 
Ber.  d.  deutsch.  chem.  Gesellsch.,  Berlin,  1889,  p.  2711). 

§  Hoppe-Seyler  and  Araki,  Zeit.  f.  physiol.  Chem.,  1895,  vol.  xx.,  p.  365. 
Osborne,  Proc.  Phys.  Soc.*  1901,  p.  xlix  ;  Jour,  of  Phys.,  vol.  xxvi. 


38  THE  EXTRACTIVES  AND  SALTS  OF  MUSCLE     [LECT. 

fermentation  lactic  acid  which  occurs  in  milk  when  it  turns  sour. 
It  has  been  found  in  small  quantities  in  muscle.* 

(ft)  Dextro-rotatory  lactic  acid.  This  is  paralactic  or  sarco- 
lactic  acid,  the  lactic  acid  par  excellence  of  muscle.  It  is  also 
found  in  the  blood,  especially  after  muscular  activity.  It  is 
found  in  the  urine  after  muscular  activity,  during  diminution 
of  oxidation  processes,  in  phosphorus  poisoning,  and  after 
extirpation  of  the  liver.  The  acidity  which  develops  after  death 
in  many  other  tissues!  and  organs  is  chiefly  due  to  the  same 
acid. 

(c]  Laevo-rotatory  lactic  acid.  This  is  produced  by  the 
fermentation  of  cane  sugar  by  certain  kinds  of  bacilli,  but  very 
little  is  known  about  it  at  present. 

In  all  cases  where  three  isomerides  exist,  as  in  the  present 
case — one  optically  inactive,  one  dextro-rotatory,  and  the  third 
laevo-rotatory — it  should  be  understood  that,  strictly  speaking, 
there  are  only  two  isomerides,  one  dextro-,  the  other 
laevo-  rotatory,  the  third  or  inactive  variety  being  a  compound  of 
the  other  two.  This  was  first  shown  by  Pasteur  in  connection 
with  racemic  acid,  which  is  optically  inactive ;  by  appropriate 
methods  of  crystallisation,  it  can  be  separated  into  two  varieties 
of  tartaric  acid,  one  dextro-rotatory,  the  other  laevo-rotatory. 

Another  interesting  method  of  separating  an  optically 
inactive  material  into  its  optically  active  components  may  be 
described  as  a  biological  method.  It  consists  in  allowing  such 
moulds  as  Penicillium  glaucum  to  grow  in  solutions  of  the 
inactive  compound ;  one  only  of  its  active  components  is 
destroyed  by  the  mould,  the  other  being  left  intact 

The  mode  of  formation  of  lactic  acid  in  muscles  has  been 
the  subject  of  numerous  researches ;  the  acid  has  been  identified 
as  sarcolactic  acid-  by  Berzelius,  du  Bois  Reymond,  Kiihne, 
Heidenhain,  and  many  others.  Its  detection  in  an  ethereal 
extract  by  means  of  Uffelmann's  reaction  t  we  have  already 
studied  (p.  6).  It  is  found  not  only  after  death,  but  also  on 
activity  during  life ;  it  is  doubtless  one  of  the  products  the 

*  Heintz,  Ann.  d.  Chem.,  1871,  vol.  clvii.,  p.  314. 

t  A  dilute  solution  of  ferric  chloride  and  carbolic  acid,  which  is  violet,  is 
turned  yellow. 


iv.]  ORIGIN  OF  SARCOLACTIC  ACID  39 

accumulation  of  which  produces  fatigue,  a  subject  we  shall  have 
to  return  to  when  we  are  studying  the  nervous  system. 

In  contrasting  together  the  different  kinds  of  muscle,  Gleiss  * 
finds  that  the  slowly  contracting  red  muscles  of  the  rabbit,  or 
the  very  slowly  contracting  muscles  of  the  tortoise,  produce  acid 
less  rapidly  than  ordinary  voluntary  muscles. 

Weyl  and  Seitler  t  were  the  first  to  point  out  that  the  increase  of  acidity 
may  be,  at  least  in  part,  due  to  acid  potassium  phosphate  produced  from  the 
alkaline  phosphate  by  the  development  of  new  phosphoric  acid  from  organic 
compounds  like  lecithin  and  nuclein.  Rohmann  \  minimises  altogether  the 
part  played  by  lactic  acid  in  the  rise  of  acidity  ;  but  the  more  recent  work 
of  v.  Fiirth  and  of  Osborne  leaves  no  reasonable  doubt  that  lactic  acid  is 
one  if  not  the  chief  cause  of  the  increased  acidity. 

With  regard  to  the  origin  of  sarcolactic  acid,  O.  Nasse 
believed  it  to  come  from  the  carbohydrates  in  the  muscle.  This 
is,  of  course,  the  simplest  view  to  take,  and  it  is  supported  by 
some  work  of  Ekunina.§  Many  facts,  however,  do  not  fit  in 
with  this  explanation  ;  for  instance,  if  the  lactic  acid  originated 
from  sugar  and  glycogen,  we  should  expect  to  find  the  same 
variety  of  the  acid  that  is  found  in  milk.  The  view  very 
generally  held  is  that  the  acid  arises  from  the  decomposition 
of  complex  molecules,  of  which  proteid  forms  a  part.  It  is  quite 
possible  that  the  lactic  acid  may  originate  in  both  ways,  and 
that  the  small  quantity  of  fermentation  lactic  acid  in  the  muscle 
may  have  a  carbohydrate  source. 

The  idea  that  the  acid  has  a  proteid  origin  was  mooted  by 
Kiihne  ||  in  some  of  his  earliest  observations  ;  he  showed  that 
not  only  is  the  acid  formed  during  rigor  mortis,  but  also  during 
the  heat  coagulation  of  myosin.  Bohm  IF  supported  the  proteid 
origin  of  sarcolactic  acid,  and  his  view  was  endorsed  by  Hoppe- 
Seyl.er.**  Some  of  my  own  experiments,  showing  the  develop- 

*  Pfliiger's  Archiv,  vol.  xli.,  p.  69. 

t  Zeit.  f.  physiol.  Chem.,  vol.  vi.,  p.  557. 

I  Pfliiger's  Archiv,  vol.  Iv.,  p.  589. 

§  Jour.  f.  prakt.  Chem.,  N.F.,  vol.  xx. 

||  Arck.f.  Anat.  u.  Phys.,  1859,  p.  795. 

*{  Pfliiger's  Archiv,  vol.  xxiii.,  p.  44  ;  vol.  xlvi.,  p.  265. 
**  Phys.  Chem.,  pp.  666,  667. 


40  TftE  EXTRACTIVES  AND  SALTS  OF  MUSCLE 

ment  of  acid  during  the  coagulation  of  pure  myosin,*  and 
Latham's  theoretical  views  j-  on  the  constitution  of  the  proteid 
molecule,  tend  in  the  same  direction.  Araki  {  found  that  the 
diminution  of  oxidation  in  the  body,  such  as  is  produced  by 
the  inhalation  of  carbonic  oxide,  leads  to  the  appearance  of 
sarcolactic  acid  (and  sometimes  sugar  and  albumin)  in  the  urine. 
This  is  accompanied  by  increase  in  proteid  katabolism,  and 
this  again,  as  Hammarsten  §  points  out,  is  in  favour  of  the  same 
view. 

It  will  thus  be  seen  that  the  bulk  of  authority  is  in  favour 
of  the  theory  that  the  lactic  acid  of  muscle  has  in  the  main  a 
proteid  origin. 

The  simultaneous  production  of  the  acid,  and  the  occurrence  of  rigor 
mortis,  have  led  some  investigators  ||  to  consider  that  the  first  is  the  cause  of 
the  second  phenomenon.  This  implies  that  the  acid  has  a  non-proteid 
origin  which  is  against  the  mass  of  evidence.  I  think  we  may  admit  that 
the  acidity  is  favourable  to  the  development  of  rigor,  without  admitting 
it  to  be  the  essential  cause.  The  development  of  acid,  and  the  formation 
-of  the  muscle  clot,  have  in  my  opinion  no  causal  relationship  between 
them  ;  both  are  the  result  of  changes  in  the  myosinogen  molecule. 

The  Nitrogenous  Extractives 

These  are  more  numerous  than  the  non-nitrogenous.  In 
connection  with  some  we  know  but  little  of  their  chemical 
composition,  and  still  less  of  their  physiological  importance. 

Thus  taurine  occurs  in  small  quantity  in  the  muscles  of 
horses,  fishes,  and  molluscs ;  glycine  is  also  found  in  molluscan 
muscle.  Protic  acid  is  a  substance  of  doubtful  nature,  also 
found  in  fishes'  muscle.  Lecithin  is  present  in  small  amount, 

*  Jour,  of  Phys.,  1887,  vol.  viii.,  p.  134.  Although  these  results  are 
criticised  by  v.  Fiirth,  they  have  been  confirmed  by  Stewart  and  Sollmann, 
Jour,  of  Phys.,  1899,  vol.  xxiv.,  p.  450. 

t  Brit.  Med.Jour.,  1886,  vol.  i.,  p.  630. 

\  Zeit.  f.  physiol.  Chem.,  vols.  xv ,  xvi.,  xvii.,  and  xix. 

§  Phys.  Chem.)  third  German  edition,  p.  332. 

||  For  instance,  Catherine  Schipiloff,  Centralbl.f.  d.  med.  Wiss.,  1882,  p. 
291.  She  found  that  injection  of  sarcolactic  and  other  weak  acids  into  the 
blood  stream  causes  a  condition  of  rigor  in  the  muscles.  This  has  been 
confirmed  by  O shorn e.  loc.  cit. 


iv.]  UREA  IN  MUSCLE  41 

but  whether  it  is  an  integral  component  of  the  muscle  itself,  or 
due  to  the  adherent  nerves,  is  doubtful.  With  the  lecithin  are 
small  quantities  of  cholesterin.  Inosinic  acid  was  first  described  _ 
by  Liebig,  who  gave  it  the  formula  C10H14N4On.  Haiser  * 
finds,  however,  that  it  contains  phosphorus,  and  ascribes  to  it 
the  formula  C10H13N4PO8.  It  is  probably  related  to  the  phos- 
phocarnic  acid  to  be  described  presently,  but  nothing  certain 
is  really  known  about  it.  Carnine  is  a  crystalline  base 
(C7H8N4O3+H2O)  originally  found  by  Weidel  in  American 
meat  extracts,  but  since  found  in  the  flesh  of  many  animals. 
It  is  probably  related  to  the  members  of  the  purine  family, 
but  again  we  have  no  certain  knowledge.  The  list  of 
imperfectly  understood  substances  has  been  recently  increased 
by  the  addition  of  a  base  called  carnosine,\  with  the  formula 
C9H14N4O3 :  it  is  probably  related  to  arginine.  The  remaining 
members  of  the  group  are  more  important,  and  demand  fuller 
study  ;  to  these  we  now  pass. 

Urea  in  Muscle 

There  can  be  but  little  doubt  that  muscular  tissue,  being  our 
most  abundant  tissue,  is  the  ultimate  source  of  most  of  the 
nitrogenous  waste  that  leaves  the  body  as  urea.  The  final 
stages  in  the  synthesis  of  urea  occur,  as  you  all  know,  in  the 
liver ;  probably  what  leaves  the  muscles  is  discharged  as 
ammonia,  which,  uniting  with  carbon  dioxide  in  the  blood, 
forms  ammonium  carbamate  or  carbonate.  The  large  subject^- 
of  nitrogenous  katabolism  thus  opened  up  is  one  of  immense 
importance,  and  might  easily  occupy  us  for  the  remainder  of 
this  course  of  lectures ;  I  must,  however,  reluctantly  pass 
it  by. 

Until  quite  a  few  years  ago,  it  was  generally  stated  that 
muscle  itself  contains  no  urea,  or  only  traces  ;  creatine,  the  next 
substance  on  our  list,  was  considered  to  represent  it,  and  as  we 
shall  find,  urea  can  be  obtained  from  creatine  in  the  laboratory. 
The  statement  concerning  urea  was  partly  due  to  imperfect 

*  Monatsheftf,  Chemie,  1895,  vol.  xvi.,  p    190. 

t  Gulewitsch  and  Amiradzbi,  Zeit.  f.  physiol,  Chetn.,  vol.  xxx.,  p.  565. 


42  THE  EXTRACTIVES  AND  SALTS  OF  MUSCLE     [LECT. 

methods  of  analysis.  Comparatively  early  it  was  shown  that 
urea  can  be  obtained  in  fair  abundance  from  the  muscles  of 
certain  animals,  for  instance,  arthropods.*  Then  many  years 
ago  Stadeler  and  Frerichs  f  found  that  the  organs,  including  the 
muscles  of  selachian  fishes,  are  rich  in  urea.  Krukenberg  J  and 
Schrceder  §  confirmed  this.  In  two  varieties  of  dogfish,  the 
mean  percentage  of  urea  in  the  blood  was  2.61,  in  muscle 
1.95,  and  in  the  liver  1.36.  Schrceder  explains  this  by  the 
fact  that  the  kidneys  are  sluggish  in  these  animals.  The 
amount  of  urea  in  these  muscles,  moreover,  is  not  modified 
by  extirpation  of  the  liver. 

By  a  new  method,  SchondorfT||  has  been  able  to  satisfactorily 
establish  the  existence  of  a  small  quantity  of  urea  in  the  muscles 
of  mammals  (0.07  to  0.2  per  cent). 

Creatine  and  Creatinine 

Creatine  can  be  crystallised  out  by  evaporating  aqueous 
extracts  of  meat,  from  which  proteids  and  salts  have  been 
previously  removed.  It  can,  for  instance,  be  readily  obtained 
from  the  meat  extracts  of  commerce.  You  will  remember  the 
red  fluid  obtained  from  pigeon's  muscle  by  the  ether  method, 
in  which  at  my  last  lecture  I  showed  you  the  spectrum  of 
myohaematin.  I  have  allowed  some  of  this  to  dry  at  room 
temperature  in  a  watch  glass.  I  will  pass  it  round,  and  you  will 
see  the  formation  in  it  of  some  very  well-formed  crystals  of 
creatine. 

Creatine  is  closely  related  to  another  basic  substance  called 

*  Krukenberg,  Vergleich.phys.  Vortrage,  1886,  p.  314. 

t  Jour.  f.  prakt.  Chem.,  1858,  vol.  Ixxiii.,  p.  48  ;  vol.  Ixxvi.,  p.  58. 

\  Loc.  cit.,  p.  314. 

§  Zeit.  f.  physiol.  Chem.,  1890,  vol.  xiv.,  p.  576. 

||  Pfliiger's  Archiv,  1895,  vol.  Ixii.,  p.  i  and  p.  332.  Although  SchondorfiPs 
methods  and  results  were  adversely  criticised  by  such  an  experienced 
chemist  as  Nencki  (Arch.  f.  exper.  Path.  Pharm.,  1895,  vol.  xxxvi.,  p.  395), 
Schondorff  very  successfully  met  these  criticisms  (Pfliiger's  Archiv,  1899, 
vol.  Ixxiv.,  p.  307),  and  his  results  have  since  been  confirmed  by  Kaufmann 
(Arch,  de  Phys.  norm,  et  path.,  ser.  5,  tome  vi.)  and  Brunton-Blaikie 
(Jour,  of  Phys.,  1899,  vol.  xxiii.,  Suppl.,  p.  44). 


IVJ  GREAT  IN  E  AND  CRE  ATI  NINE  43 

creatinine,  the  empirical   difference  between  the  two   being  a 
molecule  of  water,  as  shown  in  the  following  equation  :— 
C4H7N30  +  H20   =   C4H9N302. 

[Creatinine.]  [Creatine.] 

According  to  Voit,  the  quantity  of  creatine  in  the  voluntary 
muscles  varies  from  0.2  to  0.3  per  cent.  This  increases  during 
starvation.  Involuntary  muscle  contains  less  than  voluntary 

muscle. 

The  compound  which  zinc  chloride  forms  with  creatinine  is 
generally  used  for  isolating  this  substance  in  urine  and  other 
places  where  it  occurs.     My  own  experience  with  this  method 
has  shown  it  is  uncertain  for  quantitative  purposes.     Perhaps 
this  accounts  for  different  results  obtained  by  different  observers  ; 
thus    Neubauer   denies   the    existence    of    creatinine   in   fresh 
muscle   altogether.     Voit  and   others  say    it   increases   during 
muscular  activity,  whilst  Nawrocki  states  this  is  not  so.     Much 
more   trustworthy   results  are  obtained  by   the   use  of  G.   S. 
Johnson's  method,  in  which  he  precipitates  the  creatinine  as  a 
compound  of  mercury.*     This  method  received   the   powerful 
recommendation  of  Hoppe-Seyler  ;  f  it  may  be  used  to  identify 
creatinine  when   present   in  small   quantities;    thus  my    pupil, 
P.  C.  Colls,}  was  able  to  detect  it  in  the  blood.     It  is  quite  easy 
to  detect  creatinine  in  meat  extracts  by  this  method.     In  some 
meat  extracts  examined  by  Johnson,  and  later  by  Kemmerich,§ 
the  unexpected  result  was  obtained  that  creatinine  was  more 
abundant   than  creatine.     M6rner,||   however,  has  proved   that 
this  is  not  the  normal  state  of  things ;  if  antiseptics  are  used  to 
prevent   any    putrefaction,  creatine   is   found   to  be  the   more 
abundant  of  the  two. 

In  addition  to  creatine  and  creatinine,  other  basic  substances,  named 
xantho-creatinine  (C5H10N4O),  cruso-creatinine  (C5H8N4O),  amphi -creatine 
(C9H19N704),  and  pseudo-xanthine  (C4H5N5O),  are  stated  by  GautierU  to 

*  Proc,  Roy.  Soc.,  vol.  xlii.,  pp.  365>  493  5  vo1-  !•»  P-  28- 

t  Handb.  d.  phys.  chem.  Anal,  1893,  seventh  edition,  p.  142. 

\  Jour,  of  Phys.,  1896,  vol.  xx.,  p.  107. 

§   Zeit.fi physiol.  Chem.,  1894,  vol.  xviii.,  p.  409 

I)   Du  Bois'  Archi-u,  1898,  p.  266. 

Maly's  Jahresbericht,  vol.  xxii.,  p.  335- 


44  THE  EXTRACTIVES  AND  SALTS  OF  MUSCLE     [LECT. 

occur  in  small  quantities.  In  fishes'  muscle  Thesen*  describes  an  iso- 
creatinine.  All  these  are,  however,  at  present  mostly  in  the  region  of  the 
unknown. 

The  main  physiological  interest  of  creatine  and  creatinine 
centres  around  their  relationship  to  nitrogenous  metabolism. 
By  chemical  means  creatine  can  be  decomposed  into  sarcosine 
or  methyl  amino-acetic  acid  and  urea,  as  shown  in  the  following 
equation  :  — 


C4H9N3O2  +  H2O   =   NH.CH3.CH2.COOH  +  CON2H4. 

[Creatine.]  [Sarcosine.]  [Urea.] 

It  is  quite  possible  that  this  may  occur  in  the  body,  though  we 
are  not  always  justified  in  concluding  that  the  decompositions  of 
the  laboratory  are  comparable  to  those  taking  place  during 
metabolism.  We  are  justified  in  assuming  this  sceptical  attitude 
because  injection  of  creatine  into  the  blood  stream  of  an  animal 
leads  to  the  increase  in  the  urine,  not  of  urea  but  of  creatinine. 

Most  of  the  creatinine  of  the  urine  is  derived  from  the 
creatine  contained  in  the  meat  of  the  food.  There  is,  however,  a 
small  amount  of  creatinine  in  the  urine,  even  during  starvation, 
which  appears  to  represent  a  small  percentage  of  creatine  from 
the  muscles.  The  fate  of  the  muscular  creatine  is  one  of  the 
many  puzzling  problems  in  relation  to  the  general  question  of 
nitrogenous  metabolism  which  I  promised  to  avoid.  The  small 
amount  of  creatinine  in  the  urine  apart  from  that  which  originates 
from  the  creatine  of  the  food  will  not  account  for  it  all.  I  think 
we  may  fairly  conclude  that  the  liver  forms  urea  from  simple 
ammonium  compounds,  and  so  we  must  assume  that  the  most  of 
the  creatine  is  broken  up  into  ammonia  before  it  leaves  the 
muscles. 

Uric  Acid,  Xanthine,  and  Hypoxanthine 

These  three  substances  are  found  only  in  small  quanti- 
ties ;  the  numbers  given  are  xanthine,  0.0026  ;  hypoxanthine, 
0.022-0.026  per  cent.  ;  uric  acid,  traces.  Uric  acid  is,  however, 
more  abundant  in  reptilian  muscle. 

These   three   substances   are    important    members    of    the 

*  Zeit.f.  physiol.  Chem.,  vol.  xxiv.,  p.  i. 


iv.]  PUR1NE  SUBSTANCES  IN  MUSCLE  45 

purine    family,    and   their    names   in    this    connection    are    as 
follows : — 

Hypoxanthine        ....     C5H4N4O  .  Oxypurine. 

Xanthine C5H4N4O2  Dioxypurine. 

Uric  Acid C5H4N4O3  Trioxypurine, 

Their  formulae  sufficiently  indicate  their  close  connection. 

The  interest  of  these  substances  is  derived  in  the  first 
instance  from  their  origin  ;  they  are  the  products  of  nuclein^ 
metabolism.  In  the  second  place,  they  have  a  pathological 
interest,  and  they  would  lead  us,  if  we  were  not  careful  to  keep  to 
the  main  track,  into  the  pathological  side-issue  of  the  causation 
of  gout  and  allied  disorders.  I  \vill  be  content  to  state  the 
following  main  conclusions  arrived  at  in  connection  with  this 
question. 

1.  The  synthetic  formation  of  uric  acid  from  ammonia  and 
lactic  acid,  which  is  so  important  in  birds,  occurs  in  mammals  to 
a  slight  extent  only. 

2.  The  most  important  origin  of  uric  acid  in  mammals  is  by 
decomposition  of  nucleinrand  oxidation  of  the  purine  bases  so 
liberated. 

3.  Certain   forms    of  diet   increase  uric  acid  formation  by 
leading  to  an  increase  of  nuclear  metabolism.     This  is  indicated 
in  many  cases  by  increase  of  the  leucocytes,  and,  consequently, 
increase  in  the  metabolism  of  their  nuclei.     Although   special 
attention  has  been  directed  to  the  nuclei  of  leucocytes  because 
they  are  so  readily  examined  during  life,  it  must  be  remembered 
that  nuclein  metabolism  in  all  cells  may  contribute  to  uric  acid 
formation. 

4.  Certain  forms  of  diet  increase  the  uric  acid  in  the  urine 
and  body  generally,  because  they  themselves  contain  nuclein  or 
nuclein   derivatives   in    abundance.     Sweetbread  and  liver  are 
typical  instances  of  this  sort  of  food. 

5.  Uric  acid  which  comes  directly  from  nuclein  or  purine 
substances    in   the   food    may   be   conveniently   spoken   of    as 
exogenous :    that    which    arises    from    metabolism     is     termed  . 
endogenous  (Burian  and  Schur). 

6.  Meat  diet  causes  an  increase  in  uric  acid,  partly  because 


46  THE  EXTRACTIVES  AND  SALTS  OF  MUSCLE     [LECT. 

it  stimulates  cellular  (e.g.  leucocytic)  activity,  but  mainly  because 
it  increases  the  exogenous  uric  acid  from  the  purine  bases, 
especially  the  hypoxanthine  which  it  contains. 

Carnic  Acid 

Fleischsaure  (Carnic  acid)  is  the  name  given  by  Siegfried* 
to  a  constituent  of  muscle,  to  the  discovery  of  which  he 
attributed  great  importance.  He  first  prepared  it  from  muscle 
extracts  by  means  of  ferric  chloride  ;  the  compound  so  obtained 
is  called  carniferrin ;  this  contains  phosphorus  as  well  as  iron. 
By  means  of  baryta  water,  carnic  acid  (C10H15N3O5)  was 
separated  out  from  it.  In  muscle,  carnic  acid  is  combined  with 
phosphorus  to  form  phospho-carnic  acid  or  nucleon.  The 
first  startling  announcement  made  by  Siegfried  was  that  carnic 
acid  is  identical  with  Kiihne's  antipeptone.  Balke,f  working 
under  Siegfried's  supervision,  subsequently  prepared  compounds 
and  derivatives  of  this  material,  which  need  hardly  concern  us 
considering  the  subsequent  history  of  the  investigation.  On 
decomposition  it  yields  succinic  acid,  sarcolactic  acid,  a  reducing 
carbohydrate,  and  other  substances.  This  was  followed  by  work 
by  KriigerJ  and  what  we  may  call  a  second  startling  announce- 
ment, namely,  that  on  hydrolysis  it  gives  off  carbon  dioxide. 
He,  therefore,  looked  upon  it  as  the  material  in  muscle  which 
during  muscular  activity  gives  off  carbon  dioxide  without  the 
using  up  of  oxygen  ;  in  fact,  it  was  the  first  substance  separated 
out  from  muscle  by  chemists  which  fulfilled  the  characters 
ascribed  by  Hermann  to  his  hypothetical  inogen. 

Kruger,§  however,  in  his  subsequent  work,  discovered  that 
various  preparations  of  nucleon  gave  very  different  percentages 
of  nitrogen,  and  Siegfried  himself  was  compelled  to  admit  that 
the  varying  relationship  of  nitrogen  to  phosphorus  shows  that 
the  composition  of  nucleon  is  not  so  certain  as  he  had  at  first 
supposed.  About  the  same  time,  also,  Kutscher||  proved  that 

*  Ber.  d.  deutsch.  chem.  Gesellsch.,   1894,  vol.  xxviii.,  p.   2762  ;    Zeit.  f. 
physiol.  Chem.,  vol.  xxi.,  p.  360. 

t  Zeit.  f.  physiol.  Chem.,  1896,  vol.  xxi.,  p.  380  ;  vol.  xxii.,  p.  248. 
J  Ibid.,  vol.  xxii.,  p.  95.  $  Ibid.,  vol.  xxviii.,  p.  530. 

||  Ibid.,  vol.  xxvi.,  p.  no. 


iv.]  SALTS  OF  MUSCLE  47 

antipeptone,  although  it  is  not  a  true  peptone,  is  nevertheless 
not  a  chemical  unit  (carnic  acid),  as  alleged  by  Siegfried,  but  a 
mixture  of  various  substances  of  which  he  separated  out  aspartic 
acid,  and  the  hexone  bases  histidine  and  arginine. 

Siegfried's  work  has  the  merit  that  it  was  one  of  the  earliest 
to  shake  the  foundations  on  which  Kiihne's  theory  of  anti-  and 
hemi-  products  of  digestive  proteolysis  was  built.  He  has  also 
drawn  attention  to  certain  phosphorised  constituents  of  muscle 
which  had  hitherto  escaped  attention.  He  has,  however,  failed 
to  prove  that  nucleon  is  a  chemical  individual,  and  also  that  it 
has  the  importance  and  interest  which  he  at  first  considered  it  to 
possess. 

The  Inorganic  Salts  of  Muscle 

The  total  ash  varies  from  I  to  1.5  per  cent.  The  following 
analyses  are  by  Bunge*  :  — 

In  parts  per  1000. 


K2O       . 

.     4.654 

4.160 

Na2O     . 

.    0.770 

0.811 

CaO 

.     0.086 

0.072 

MgO     .." 

...'..     .        .    0.412 

*     /  * 

0.381 

Fe2O3    . 

.       .  -v  .       .    0.057 

... 

P9O* 

4.644 

4.<;8o 

Cl. 

...     .    0.672 

*T*  J  wvx 

0.700 

SO, 

0.100 

J.  Katz  j-  gives  the  following  numbers  from  the  examination 
of  the  flesh  of  a  large  number  of  animals  ;  they  represent  the 
minimum  and  maximum  in  1000  parts  of  fresh  flesh. 

K       .     '-.-.        .        .        .        .        .  2.4    to  4.6 

Na     .        .        .        .  ^   /      .        .  0.3    to  1.5 

Fe      .   -     .      \.        .        .        »        .  0.04  to  0.25 

Ca      .        ....        *        .        .  0.02  to  0.39 

Mg    .        .        .   -"    .        .    .    .   .  ,  .  0.18  to  0.37 

P  (from  phosphates)  .         .         .        .  1.22  to  2.04 

(from  lecithin)         ....  0.13  to  0.48 

(from  nuclein)        »;       .        .         .  0.09  to  0.32 

Cl       .......  0.32  to  0.8 

*  Zeit.f.physwl.  Chem.,  vol.  ix.,  p.  60. 
+  Pfliiger's  Archiv^  1896,  vol.  Ixiii.,  p.  I. 


48        THE  EXTRACTIVES  AND  SALTS  OF  MUSCLE    [LECT.  iv. 

We  at  once  note  the  predominance  of  potash  *  among  the 
bases,  and  of  phosphoric  acid  among  the  acids. 

I  will  conclude  with  a  passing  allusion  to  the  importance  of 
mineral  constituents  in  the  causation  of  muscular  contraction. 
Many  years  ago,  Ringer  showed  that  contractile  tissues  continue 
to  manifest  their  activity  in  pure  solutions  of  salts  correspond- 
ing in  proportion  to  those  found  in  the  blood.  This  question 
has  more  recently  been  taken  up  in  America  by  Howell  at 
Baltimore,  and  Loeb  at  Chicago  and  California.  Since  the 
introduction  of  the  ionic  theory,  these  results  of  Ringer's  have 
been  interpreted  as  ionic  action.  Contractile  tissues  will  not 
contract  in  pure  solutions  of  non-electrolytes  (sugar,  urea, 
albumin).  But  different  contractile  tissues  differ  in  the  nature 
of  the  ions  which  are  most  favourable  stimuli.  Loeb  divides 
them  into  three  classes:  (i)  These  which  produce  such  con- 
tractions ;  of  these  the  most  efficacious  is  Na.  (2)  Those  which 
retard  or  inhibit  rhythmical  contractions  ;  for  instance,  Ca  and 
K.  (3)  Those  which  act  catalytically  ;  that  is,  they  accelerate 
the  action  of  Na,  though  they  do  not  themselves  produce 
rhythmical  contractions  directly ;  for  instance,  H  and  OFT  In 
spite  of  the  antagonistic  effect  of  Ca,  a  certain  minimal  amount 
of  it  must  be  present  if  contractions  are  to  continue  for  any 
length  of  time.  We  have  already  seen  that  the  influence  of 
calcium,  which  is  so  important  in  blood-clotting  and  milk- 
curdling,  is  a  doubtful  factor  in  the  production  of  muscle-clotting 
or  rigor  mortis.  There  is,  however,  no  doubt  as  to  its  necessity 
in  muscular  contraction.  Ions  produce  contraction  because  they 
affect  either  the  physical  condition  of  the  colloidal  substances 
(proteid,  etc.),  in  protoplasm,  or  the  rapidity  of  chemical 
processes. 

*  Prof.  A.  B.  Macallum  has  recently  introduced  a  micro-chemical  test  for 
potassium  in  tissues.  It  consists  in  adding  to  the  fresh  tissue  a  mixture  of 
the  nitrites  of  sodium  and  cobalt ;  an  abundant  precipitate  of  yellow  crystals 
is  the 'result.  If  the  amount  of  potassium  is  small,  there  is  only  a  yellow 
colour  ;  this  is  turned  black  (cobalt  sulphide)  on  the  addition  of  ammonium 
sulphide.  The  black  particles  seen  form  a  very  delicate  test.  In  a  muscular 
fibre  the  potassium  is  limited  to  the  dark  bands.  His  paper  will  shortly 
appear  in  the  Journal  of  Physiology. 


LECTURE   V 

CHEMICAL   CHANGES   ACCOMPANYING   THE   CONTRACTION 
OF   MUSCLE.      CHEMISTRY  OF   TENDON 

WE  have  now  to  attempt  an  answer  to  what  is,  after  all,  the 
most  important  question  in  relation  to  muscular  activity, 
namely,  what  are  the  chemical  changes  which  accompany  its 
contraction.  There  is  no  doubt  that  it  is  the  chemical  change 
which  underlies  the  other  manifestations  of  muscular  activity ; 
the  heat  produced,  and  the  electrical  current  of  action  being 
secondary  to  this.  In  the  transformation  of  the  energy  of 
chemical  affinity,  some  reappears  as  heat,  and  a  variable  but 
comparatively  small  fraction  as  muscular  work. 

It  is  in  order  to  answer  this  question  that  up  to  this  point 
we  have  been  gathering  materials.  We  are  somewhat  in  the 
position  of  an  engineer  who  has  been  collecting  the  individual 
parts  of  a  machine,  and  learning  the  uses  of  each  before  he 
proceeds  to  investigate  the  way  in  which  the  machine  works  as 
a  whole.  It  is  just  here  we  have  to  confess  the  imperfection  of 
our  knowledge ;  we  have,  it  is  true,  a  number  of  facts  to  rely 
upon  ;  we  have  noted,  however,  in  going  along,  how  many  more 
facts  we  ought  to  have  before  we  can  found  trustworthy  and 
workable  theories. 

While  a  muscle  is  at  rest,  we  do  not  mean  it  is  absolutely 
inactive  ;  we  know,  for  instance,  that  it  possesses  that  small 
amount  of  contraction  which  is  technically  known  as  "  tonus." 
There  is  also  what  we  may  call  "  chemical  tonus  " ;  the  evidence  . 
of  this  is  that  the  blood  leaving  muscles  which  are  not 
contracting  is  nevertheless  venous,  and  heat  production  is 
occurring  in  muscles  which  are  in  repose.  In  all  probability, 
49  D 


50  CHEMICAL  CHANGES  ON  CONTRACTION          [LECT. 

the  chemical  changes  that  occur  during  contraction  are  similar 
in  kind  to  those  which  occur  during  so-called  rest ;  there  is  a 
sudden  exaggeration  of  the  normal  "chemical  tonus"  of  the 
tissue,  and  an  explosive  liberation  of  energy. 

The  first  trustworthy  observations  in  this  connection  were 
made  by  Helmholtz  ;  he  showed  that  during  contraction  the 
extractives  soluble  in  alcohol  increase,  and  those  soluble  in 
water  decrease.  This  is  chiefly  explicable  by  the  disappearance 
of  glycogen,  and  the  appearance  of  sugar  and  lactic  acid. 
Research  has  shown  that  on  contraction,  proteid  katabolism  is 

-somewhat  increased;  still  in  a  normally  nourished  muscle  the 
main  work  falls  on  the  non-nitrogenous  part  of  the  muscle,  as  is 
shown  by  the  immediate  increase  in  the  amount  of  carbon 

-dioxide  which  leaves  it.     (See  Lecture  IV). 

In  a  general  way  we  may  speak  of  the  essential  chemical 
process  as  one  of  oxidation,  in  that  oxidised  products  are  dis- 
charged ;  but  the  oxidation  is  not  of  that  simple  kind  which 
occurs  when  oxygen  combines  with  the  combustible  material  in 
a  candle.  Oxygen  in  muscle,  as  in  other  tissues,  is  not  only  of 
importance  for  the  formation  of  katabolic  products,  but  is  of 
even  greater  use  in  anabolism ;  it  assists  in  the  preliminary 
building  up  of  complex  materials  within  the  living  molecules. 
Hermann's  celebrated  inogen  theory  was  the  outcome  of  such 
a  conception  ;  there  is  no  oxygen  among  the  gases  which  he 
extracted  from  muscle,  and  a  muscle  will  continue  to  contract 
and  give  off  carbon  dioxide  in  an  atmosphere  free  from  oxygen. 
The  oxygen  used  in  the  formation  of  this  carbonic  acid  must 
therefore  have  been  held  in  some  compound  within  the  tissue, 
and  this  hypothetical  body  was  termed  inogen.  The  theory  in 
all  its  details  no  longer  stands ;  yet  it  embodies  the  undoubted 
•fact  that  the  role  of  oxygen  is  more  in  the  constructive  than  in 

'the  destructive  phase  of  metabolism.     The  term  oxygenation 

'better  expresses  this  than  oxidation.  The  difficulties  surround- 
ing the  question  have  led  some  physiologists  to  postulate  the 
existence  of  oxidases,  or  oxygen  carrying  ferments.  I  regard  it 
as  very  questionable  whether  they  are  really  enzymes,  but  if 
such  materials  exist,  it  appears  probable  that  their  usefulness  is 
not  in  promoting  oxidation,  a  katabolic  change,  but  rather  what 


{  UNIVERSITY  j 


v.]  HERMANN'S  INOGEN  TH. 

we  have  just  called  oxygenation,  that  is  the  building  of  the 
oxygen  into  the  living  molecules. 

Hermann's  theory  of  muscular  contraction  further  assumes 
that  the  change  is  similar  in  kind  to  that  which  occurs  on  death, 
though  less  in  degree.  On  death  he  considered  that  inogen  is 
split  into  carbon  dioxide,  sarcolactic  acid,  and  myosin.  He  thus 
throws  in  his  lot  with  those  who  hold  that  sarcolactic  acid  comes 
from  a  proteid  complex  and  not  from  carbohydrate.  We  have, 
however,  no  proof  that  any  formation  of  a  clot  of  myosin  occurs 
in  the  contraction  of  living  muscle ;  in  fact,  the  observations  on 
the  extensibility  of  living  contracted  muscle,  as  compared  with 
that  of  rigored  muscles,  tend  in  exactly  the  opposite  direction. 

Hermann's  theory  put  in  another  way,  that  living  contracted 
muscle  is  muscle  on  the  road  to  death,  could  only  be  proved  by 
finding  an  exact  parallelism  in  all  details ;  the  table  I  am  going 
to  give  you  immediately  shows  in  how  many  details  the  analogy 
breaks  down.  The  theory  is  interesting  from  an  historical  point 
of  view,  and  it  also  serves  as  a  useful  peg  for  a  lecturer  to  fix  the 
students'  attention  when  speaking  of  the  similarities  in  the  two 
cases. 

Here  is  the  table  I  just  mentioned. 


Dead  Muscle  in  Rigor  Mortis. 


Living  Muscle  in  Contraction. 


1.  Muscle  shortened  and  opaque. 

2.  Carbon  dioxide  given  off. 

3.  Sarcolactic  acid  formed. 

4.  Heat  evolved  at  onset  of  rigor. 

5.  Muscle    becomes    electro-positive    to 

living  muscle. 

6.  Muscle  plasma  clots. 

7.  Extensibility  is  diminished. 

8.  Rigor  is  hindered  by  dextrose. 

9.  Whether  calcium  is  essential  is  un- 

certain. 


1.  Muscle  shortened,  but  not  opaque. 

2.  Carbon  dioxide  given  off. 

3.  Sarcolactic  acid  formed. 

4.  Heat  evolved  on  contraction. 

5.  Muscle    becomes    electro-positive    at 

commencement  of  contraction 

6.  Muscle  plasma  does  not  clot. 

7.  Extensibility  is  increased. 

8.  Contraction  is  favoured  by  dextrose. 

9.  Calcium  is  essential. 


I  think  you  will  agree  with  me  that  our  knowledge  on  this 
question  is  unsatisfactory.  It  is  so  fragmentary  ;  and  even  if  I 
could  piece  together  all  the  other  fragments  of  our  present  know- 
ledge which  I  have  omitted,  we  should  still  be  without  the  means 


52  CHEMICAL  CHANGES  ON  CONTRACTION          [LECT. 

of  producing  a  perfect  picture.  I  .will  only  trouble  you  with  one 
more  fragment,  and  this  relates  to  the  formation  of  reducing 
substances.  Resting  muscle  oxidises  pyrogallic  acid  ;  tetanised 
muscle  does  not.  A  solution  of  nitrites  passed  through  con- 
tracting muscle  is  changed  into  one  of  nitrates,  and  the  colour 
of  solutions  of  indigo  sulphate  is  altered  in  the  same  way  as  by 
reducing  agents.*  A.  Schmidt  j-  arrived  at  the  same  conclusion 
from  the  examination  of  the  venous  blood  of  tetanised  muscle  ; 
but  what  the  reducing  substances  are  that  are  produced  is  quite 
unknown.-' 

Chemistry  of  Tendon 

It  is  rather  a  sudden  jump  tp  pass  from  such  an  eminently 
living  tissue  as  muscle  to  a  comparatively  passive  material  like 
its  tendon.  The  anatomical  proximity  of  the  two  is  my  excuse 
for  doing  so. 

The  chemistry  of  the  connective  tissues  has  always  possessed 
a  great  attraction  for  me,  and  I  was  originally  led  to  take  it  up 
in  connection  with  the  question  of  myxcedema.  In  this  disease 
there  is  not  only  the  atrophy  of  the  proper  substance  of  the 
thyroid  gland,  and  various  nervous  symptoms  which  I  need  not 
go  into,  but  also  a  swollen  condition  of  the  body  which  led  to 
the  adoption  of  the  name  myxcedema.  This  name  will  probably 
not  be  altered  now,  but  it  is  nevertheless  a  misnomer.  The  idea 
in  Ord's  mind  that  the  swollen  appearance  of  the  subcutaneous 
tissues  is  due  to  excess  of  mucin  has  been  abundantly  disproved. 
There  is  only  a  slight  increase  of  mucin  in  an  early  stage  of  the 
overgrowth  of  these  tissues ;  new  connective  tissues,  as  in  the 
fcetus  and  child,  always  contain  a  relatively  large  amount  of 
intercellular  or  ground  substance,  of  which  an  important  con- 
stituent is  mucin.  In  later  stages,  when  development  of  fibres 
or  deposition  of  fat  takes  place,  this  relative  increase  of  mucin 
disappears. 

My  former  pupil,  Dr  R.  A.  Young,  took  up  at  my  suggestion 
a  number  of  other  questions  in  connection  with  the  connective 

*  Griitzner,  Pfltiger's  Archiv,  vol.  vii.,  p.  255  ;  Gscheidlen,  ibid.,  vol.  viii., 
p.  506. 

t  Sitzungsber  d.  Akad.  d.  Wisscnsch.^  Wien,  vol.  xx. 


v.]  CHEMISTRY  OF  TENDON  53 

tissues,  but  of  recent  years  the  centre  of  work  on  these  points 
has  shifted  across  the  Atlantic,  and  Dr  William  J.  Gies  is  the 
investigator  who  has  now  made  the  subject  his  own.  His 
numerous  papers  will  be  mainly  found  in  the  American  Journal 
of  Physiology. 

A  tendon,  and  we  must  restrict  ourselves  to  this,  contains 
about  63  per  cent,  of  water.  The  most  abundant  solid  is  collagen, 
the  material  of  which  the  white  fibres  are  composed.  It  is  so 
named  because  it  is  the  mother  substance  of  gelatin,  into  which 
it  is  easily  converted  by  boiling.  The  other  histological 
elements  are  the  tendon  cells  and  the  ground  substance.  This 
latter'  material  is  generally  (on  the  strength  of  the  older  observa- 
tions of  Rollett  and  Loebisch)  spoken  of  as  muco-albuminous, 
an  adjective  which  recent  work  has  shown  to  be  correct.  The 
total  amount  of  mucin  is  generally  under  I  per  cent.,  and  the 
amount  of  albumin  is  even  less.  Small  quantities  of  extractives 
(including  creatine)  and  inorganic  salts  complete  the  list. 

The  ground  substance  of  connective  tissue  was  investigated 
by  Young  in  the  vitreous  humour  of  the  eye,  and  the  Whar- 
tonian  jelly  of  the  umbilical  cord,  because  in  those  situations 
fibres  and  cells  are  at  a  minimum.  Mucin  is  a  term  which 
covers  a  number  of  substances,  all  soluble  in  dilute  alkali,  pre- 
cipitable  by  acetic  acid,  and  in  constitution  consisting  of  proteid 
combined  with  a  carbohydrate  radicle.  The  difference  in  pro- 
portion between  the  proteid  and  carbohydrate  components  prob- 
ably accounts  for  the  differences  between  the  members  of  the 
mucin  group.  The  carbohydrate  material  was  at  one  time 
called  u  animal  gum,"  after  Landwehr ;  the  reducing  substance 
obtained  by  boiling  mucin  with  mineral  acid  is  not  sugar,  but  an 
amino-derivative,  glucosamine  (C6HnO5.  NH2),  in  many  cases 
at  any  rate. 

The  gluco-proteids  of  connective  tissue  are  now  generally 
called  mucoids,  the  term  mucin  being  restricted  to  gluco- 
proteids  of  cellular  origin,  for  instance  in  the  goblet  cells  of 
columnar  epithelium.  Though  Gies,  from  slight  discrepancies  in 
ultimate  analyses,  suspects  that  more  than  one  member  of  the 
gluco-proteid  group  is  present  in  connective  tissue,  he  has 
shown  that  the  principal  mucoid  in  tendon  (tendo-mucoid)  is 


54  CHEMISTR  Y  OF  TENDON  [LECT. 

the  same  material  as  that  which  can  be  separated  out  from  other 
forms  of  connective  tissue  (chondro-mucoid,  osseo-mucoid,  etc.). 

Young  and  Gies  agree  that  the  proteid  matter  (other  than 
the  mucoid)  contained  in  the  ground  substance  can  be  separated 
into  an  albumin  and  a  globulin.  But  the  amount  of  each  is  so 
small  that  their  characters  have  not  been  fully  worked  out ; 
moreover,  it  is  quite  probable  that  in  part  these  substances  are 
derived  from  adherent  lymph.  Their  temperature  of  heat 
coagulation  is  differently  given  by  the  two  observers,  and  this 
again  is  what  one  expects  in  experiments  in  which  dilute 
solutions  of  proteid  are  employed.  Brodie  and  Richardson,  in 
their  work  on  heat  rigor  of  muscle,  found  that  tendon  also 
shortens  very  considerably  at  a  certain  temperature,  namely,  63° 
C,  and  this  temperature  is  very  constant,  but  we  cannot  at 
present  say  which  proteid  constituent  of  tendon  is  responsible 
for  this. 

Another  question  which  we  must  answer  before  concluding 
is,  whether  tendon  contains  reticulin.  It  is  first  necessary  to 
explain  what  reticulin  is.  The  fibres  of  reticular  or  retiform 
tissue  (found  in  such  situations  as  lymphatic  glands,  or  the 
corium  of  many  mucous  membranes)  are  anatomically  con- 
tinuous with  those  of  areolar  tissue,  and  are  not  distinguishable 
from  them  on  microscopic  examination.  It  would  therefore 
have  been  remarkable,  if  they  were  demonstrated  to  be  chemi- 
cally different  from  ordinary  white  fibres.  The  statement  that 
this  is  so  was,  however,  made  by  Mall.*  At  first  he  thought 
they  were  made  of  elastin,  but  subsequently,  finding  this  was 
not  so,  he  considered  that  they  were  made  of  something  else, 
but  certainly  not  of  collagen,  because  no  gelatin  could  be 
obtained  from  them.  This  error  was  pointed  out  by  R.  A. 
Young; I  gelatin  is  obtained  from  reticular  fibres  with  great 
ease,  and  this  observation  was  subsequently  confirmed  by 
Siegfried.]:  Siegfried,  however,  stated  that,  in  addition  to  col- 

*  Anat.  Anzeiger,  1888,  vol.  iii.,  No.  14  ;  Abhandl.  d.  math.phys.  Cl.  d.k. 
Sachs.  Gesellsch.  d.  Wissensch.,  1887,  vol.  xiv.,  No.  3  ;  1891,  vol.  xvii.,  No.  4. 

t  Jour,  of  Phys.,  1892,  vol.  xiii.,  p.  332. 

|  Ueber  die  chemischen  Eigenschaften  des  reticulirten  Geiuebes. 
Habilitationschrift,  Leipzig,  1892. 


v.]  RETICULIN  55 

lagen,  the  reticular  fibres  contain  something  special,  and  separ- 
ated from  them  not  only  gelatin,  but  another  chemical  substance 
specially  resistant  to  the  action  of  reagents,  and  to  this  he  gave 
the  name  of  reticulin.  If  such  a  chemical  substance  does  exist, 
the  point  is  by  no  means  proved  that  reticular  fibres  are  different 
from  white  connective-tissue  fibres  ;  it  is  at  least  equally  pos- 
sible that  reticulin  may  be  present  in  all  white  connective-tissue 
fibres,  and  I  therefore  suggested  to  Miss  Tebb  that  she  should 
look  for  it  in  a  typical  form  of  connective  tissue,  namely,  tendon. 

Siegfried's  work  had  been  so  highly  spoken  of,  and  his 
conclusions  accepted  so  unhesitatingly  by  Dr  Gamgee,*  that 
you  may  well  imagine  that  when  Miss  Tebb  was  unable  to 
find  a  trace  of  reticulin  in  tendon,  my  disappointment  was  very 
great.  But,  instead  of  stopping  here,  this  failure  to  obtain 
reticulin  from  tendon  led  us  next  to  repeat  Siegfried's  experi- 
ments on  the  tissue  with  which  he  had  himself  worked,  namely, 
the  mucous  membrane  of  the  intestine.  I  knew  from  previous 
experience  that  the  prolonged  action  of  alcohol  on  collagen 
renders  it  very  difficult  to  convert  into  gelatin.  Miss  Tebb 
showed  the  same  to  be  true  for  ether,  though  in  a  less  degree. 
Neither  alcohol  nor  ether,  however,  have  any  appreciable  effect 
of  this  kind  on  gelatin.  Both  of  these  reagents  were  used  by 
Siegfried  for  prolonged  periods  to  extract  the  fat  from  the 
mucous  membrane.  It  was  therefore  quite  on  the  cards  that 
reticulin  was  merely  an  artifact,  and  the  conclusion  at  which 
Miss  Tebb  arrived  may  be  best  expressed  in  her  own  words : — 

"  My  main  result  is  that  reticulin  does  not  exist  either  in 
ordinary  white  fibrous  tissue  (tendon),  or  in  the  reticular  tissue 
of  the  intestinal  mucous  membrane.  Both  consist  of  fibres 
which  are  chemically  and  histologically  identical ;  the  main 
material  of  which  they  are  composed  is  the  gelatin-yielding 
substance  called  collagen. 

"  I  regard  Siegfried's  reticulin  merely  as  collagen  which  has 
been  '  coagulated '  by  the  reagents  he  employed  (especially 
alcohol  and  ether),  plus  proteid  and  nuclein  residues  of  cells. 
After  treatment  with  these  reagents,  the  conversion  into  gelatin 
is  much  more  difficult,  not  only  in  the  case  of  a  finely  stranded 
*  Phys.  Chem.,  1893,  vo^-  "•>  P-  4°3- 


56  CHEMISTRY  OF  TENDON  [LECT.  v. 

tissue  like  reticular  tissue,  but  even  in  a  dense  material  like 
tendon." 

I  can  vouch  for  the  accuracy  and  care  with  which  Miss 
Tebb  carried  out  the  investigation,  and  it  was  most  curious  to 
see,  as  I  watched  the  progress  of  the  work,  how  erroneous 
even  on  minor  points  Siegfried's  observations  had  been.  The 
two  new  substances,  carnic  acid  and  reticulin,  with  which 
Siegfried's  name  is  linked,  have  thus  both  met  with  an 
unfortunate  end.* 

We  have  now  completed  our  study  of  the  first  subject 
of  the  course,  and  the  remaining  lectures  I  have  to  deliver  will 
deal  with  some  of  the  biochemical  aspects  of  nerve  physiology. 

*  Siegfried  published  a  polemical  reply  to  Miss  Tebb  in  the  Jour,  of 
Phys.,  1902,  vol.  xxviii.,  p.  319.  He  does  not,  however,  shake  in  the  least 
the  conclusions  she  arrived  at. 


LECTURE   VI 

THE   CHEMICAL  COMPOSITION   OF   NERVOUS   TISSUES 

THE  nervous  system  has  always  a  great  fascination  for  physi- 
ologists and  physicians.  It  is  the  ruling  system  of  the  body, 
regulating  and  controlling  the  other  processes  that  occur  there. 
It  is,  moreover,  the  seat  of  mental  phenomena,  and  so  its 
investigation  appeals  to  all  those  who  have  sought  to. unravel 
the  so-called  mysteries  of  thought  and  reason.  But  neither  the 
scalpel,  the  microscope,  or  the  test-tube  have  as  yet  succeeded  in 
discovering  the  mind.  Still,  even  in  our  test-tubes  we  shall 
find  something  that  is  of  interest,  and  I  hope  also  of  practical 
importance.  We  shall  first  deal  with  the  physiological,  and 
later  with  the  pathological,  side  of  the  subject.  We  shall  therefore' 
start  with  the  general  chemical  composition  of  nervous  tissues. 

Relation  of  Water  and  Solids 

The   first   general    impression    one   derives   from  a   glance 
through  any  of  the  analytical  tables  in  the  text-books,  is  the 
great   preponderance  of  water  in   most  of  our  so-called  solid 
tissues.      Even    bone   contains    nearly    50    per   cent,   of  water,  - 
and  muscle,  as  we  have  already  seen,  contains  75  per  cent,  or . 
more. 

The  nervous  system  is  no  exception  to  this  rule.  The 
amount  of  water  varies  ;  it  is  present  in  larger  amount  in  early 
than  in  adult  life,  in  grey  than  in  white  matter,  in  the  brain 
than  in  the  spinal  cord,  in  the  spinal  cord  than  in  nerves, 


58       CHEMICAL  COMPOSITION  OF  NERVOUS  TISSUES    [LECT. 
This  is  illustrated  by  the  following  tables  : — 
Influence  of  Age  on  the  Percentage  of  Water  in  the  Brain  Tissue* 

White  Matter.        Grey  Matter. 

In  foetus          ....         87  92 

Age  20-30       ....         69  83 

Age  70-90       ....        72  84 

Percentage  of  Water  in  Different  Parts  of  the  Nervous  System  of  the  AdultA 

Grey  matter  of  brain  .  .  .  .  .  81-86 
White  matter  of  brain  ....  68-72 
Brain  as  a  whole  .  .  .  .  .81 

Spinal  cord 68-76 

Nerves        .         .         .         .         .         .         .     57-64 

Percentage  of  Water  in  Different  Parts  of  the  Nervous  System.\ 

Grey  matter  of  cerebrum      ....  83.4 

White  matter  of  cerebrum    ....  69.9 

Cerebellum 79.8 

Spinal  cord  as  a  whole          .         .         .         .71.6 

Cervical  cord 72.5 

Dorsal  cord 69.7 

Lumbar  cord         ......  72.6 

Sciatic  nerves       .    '   .    V  .        .        ..       .  65.3 

We  thus  see  that  water  is  most  abundant  in  the  grey  matter, 
or  in  those  regions  of  the  nervous  system  where  the  proportion 
of  grey  matter  is  greatest.  It  cannot  fail  to  be  a  striking  fact 
that  the  grey  matter,  the  region  which  is  most  active  and  most 
important,  contains  somewhat  less  than  17  per  cent,  of  solid 
materials. 

Specific  Gravity 

The  question  of  the  amount  of  water  is  closely  related  to 
that  of  specific  gravity.  The  specific  gravity  of  the  brain  has 
been  the  subject  of  researches  by  Bastian,  Bischof,  Danilewski, 
and  others,  but  I  only  wish  here  to  dwell  upon  one  aspect  of  the 

*  Weisbach,  Hofmanris  Lehrbuch  d.  Zoochemie,  Wien,  1876,  p.  121. 

t  Table  compiled  from  a  number  of  analyses  made  by  others. 

\  This  table  gives  the  averages  of  a  large  number  of  experiments  made 
with  the  organs  of  human  beings,  monkeys,  dogs,  and  cats,  by  myself 
(Jour,  of  Phys.)  1893,  vo^  xv->  P-  9°)- 


vi.]  SPECIFIC  GRA  V1TY  OF  BRAIN  59 

question  which  was  brought  prominently  before  the  medical 
profession,  in  an  address  on  "  Sex  in  Education,"  by  Sir  James 
Crichton  Browne,*  some  years  ago.  Among  the  differences 
between  the  brains  of  men  and  women,  Sir  James  stated  that 
he  had  found  that  the  specific  gravity  of  the  female  brain  is  less 
than  that  of  the  male  brain.  It  was,  however,  pointed  out 
in  the  correspondence  that  followed  the  publication  of  the 
address,  that  this  generalisation  rested  on  very  few  observations. 
I  have  accordingly  thought  it  advisable  to  examine  the  brains 
in  a  large  number  of  cases.  This  has  been  carried  out  in  my 
laboratory  by  R.  H.  Gompertz,  B.Scf  All  I  shall  do  here  is  to 
mention  his  main  conclusion.  He  finds  that  in  adult  men  and 
women,  who  suffered  from  no  brain  disease,  that  there  are  fairly 
wide  variations  in  both  sexes,  but  that  the  average  (1.035)  ls- 
identical  in  male  and  female,  and  that  there  is  no  founda- 
tion for  the  belief  that  the  variations  constitute  a  sexual 
difference. 

I  may  point  out  that  a  low  specific  gravity  of  the  brain  does 
not  necessarily  imply  a  poorer  quality,  for  the  part  of  the  brain 
which  is  most  important  and  most  active — the  grey  matter — has  - 
a  lower  specific  gravity  than  the  white  matter. 

If,  for  instance,  Sir  James  Crichton  Browne  had  satisfactorily 
established  his  contention  that  a  woman's  brain  is  of  lower 
specific  gravity  than  a  man's,  the  conclusion  might  have  been 
drawn  that  the  female  brain  is  richer  in  grey  matter,  and  the 
ladies  might  quite  fairly  have  argued  that  what  they  lacked  in 
quantity  was  made  up  in  quality.  The  question,  however,  is  not 
so  simple  as  this,  for,  as  Dr  Bastian  pointed  out  to  me,  an 
increase  of  association  fibres  which  doubtless  underlies  intel- 
lectual superiority  would  mean  an  increase  of  white  matter,  and 
lead  to  a  raising  of  the  specific  gravity,  Boileau  J  drew  attention, 
in  the  examination  he  made  of  the  brain  of  a  highly  gifted  man, 
not  only  to  its  great  weight,  but  also  to  its  Jiigh  specific  gravity. 
A  great  many  more  similar  cases  will  have  to  be  recorded,  before 
any  general  rule  can  be  laid  down  on  the  subject. 

*  Brit.  Med.Jour.,  1892,  vol.  i.,  p.  949. 
t  Jour,  of  Phys.,  1902,  vol.  xxvii.,  p.  459. 
\  Lancet^  1882,  vol.  ii.,  p.  485. 


60       CHEMICAL  COMPOSITION  OF  NERVOUS  TISSUES    [LECT. 


Solids  of  Nervous  Tissues 

But  coming  now  to  the  more  important  subject  of  the  solids, 
we  find  it  is  possible  to  divide  them  into  the  following  classes  : — 

(a)  Proteids.     These  comprise  a  very  considerable  percentage 
of  the  solids,  especially  in  the  grey  matter  (over  50  per  cent). 

(b)  Nuclein.     From  the  nuclei  of  the  cells. 

(c)  Neuro-keratin.     From   the  supporting  framework  (neu- 
roglia). 

(d}  Phosphorised  fats  (protagon,  lecithin,  etc.). 

(e)  Cerebrins  or  cerebrosides. 

(/)  Cholesterin. 

(^•)  Extractives.  Small  quantities  of  numerous  organic  sub- 
stances, of  which  creatine,  xanthine,  hypoxanthine,  inosite,  lactic 
acid,  uric  acid,  and  urea  have  been  identified. 

(h)  Gelatin.     From  the  adherent  connective  tissue. 

(z)  Inorganic  salts.  Of  these,  alkaline  phosphates  and 
chlorides  are  the  most  abundant,  but  the  total  ash  is  only  about 
i  per  cent* 

The  following  table  gives  some  typical  quantitative  analyses 
which  have  been  made  of  the  proportion  in  which  the  principal 
solids  occur  in  different  nervous  structures  : — 


Portion  of  Nervous 
System. 

Proteids. 

Lecithin. 

Choles- 
terin 
and  fat. 

Cerebrin. 

Neuro- 
keratin. 

Other 
organic 
matters. 

Salts. 

Grey  matter   of    ox 

brain  (Petrowsky) 

55-37 

17.24 

18.68 

0-53 

6. 

71 

1-45 

White  matter  of  ox 

brain  (ibid.') 

24.72 

9.90 

V 

51.9 

955 

3- 

34 

0-57 

Spinal  cord  (Moles-  ^\ 
chott)     .     .    .     ./ 

23.8 

75-i 

I.I 

Human  sciatic  nerve 

(Josephine  Cheva- 

lier)     

36  8 

?2.C7 

12.22 

11.30 

•3.07 

4  O 

*  It  appears  desirable  that  fresh  investigations  on  the  ash  of  nervous 
tissues  should  be  undertaken.  Macallum  has  shown  by  his  micro-chemical 
^test  (see  p.  48)  that  potassium  is  absent  from  the  nerve-cell  and  the  axon. 
It  is  present  in  the  interstitial  tissue,  and  occurs  in  curious  patches  within 
the  neurilemmal  sheath.  Another  important  generalisation  of  his  work  is 
that  potassium  is  absent  from  the  nuclei  of  all  animal  and  vegetable  cells. 


VI.] 


P  ROTE  JDS  OF  NERVOUS  TISSUES 


61 


We  are  thus  provided  at  the  outset  with  a  very  long  list  of 
substances  to  consider,  but  happily  for  you,  though  no  doubt 
unhappily  for  science,  we  know  comparatively  little  of  many  of 
these  mentioned,  or  at  least  we  are  unacquainted  with  their 
physiological  significance.  In  connection  with  the  group  of 
extractives,  for  instance,  we  can  surmise  that  they  are  mostly 
waste  products,  as  they  are  elsewhere.  In  connection  with 
cholesterin,  the  large  amount  present  of  this  remarkable  mona- 
tomic  alcohol  cannot  be  devoid  of  importance,  but  at  present  we 
cannot  even  conjecture  what  that7 importance  consists  in.  Of 
the  substances  mentioned,  those  which  will  require  further 
notice  are  the  proteids,  the  phosphorised  fats,  and  the  cere- 
brosides. 

Proteids  of  Nervous  Tissues 

The  large  amount  of  proteid  matter,  next  to  the  high  per- 
centage of  water,  is  the  most  striking  fact  in  the  preceding  table. 
The  highest  percentage  is  found  where  one  would  expect  it, 
namely,  in  the  grey  matter,  where  the  protoplasmic  structures, 
the  nerve-cells,  occur. 

The  following  table  is  a  compilation  from  my  own 
analyses  : — 


Percentage  of 

Water. 

Solids. 

proteid  in 

solids. 

Grey  matter  of  cerebrum 

83.467 

16.533 

51 

White 

69.912 

30.088 

33 

Cerebellum  . 

79.809 

20.191 

42 

Spinal  cord  as  a  whole 

71.641 

28.359 

31 

Cervical  cord 

72.529 

27.471 

3i 

Dorsal  cord  .         .    '  '.V  '  C 

69-755 

30.245 

28 

Lumbar  cord 

72.639 

27.361 

33 

Sciatic  nerves        .         .            ...-"•• 

61.316 

38.684 

29 

This  table  illustrates  the  fact  that  the  amount  of  grey  matter, 
of  water,  and  the  percentage  of  proteid  in  the  solids,  vary  directly 
the  one  with  the  others.  This  is  very  well  seen  in  the  different 
regions  of  the  spinal  cord.  The  percentage  of  proteid  matter  in 
the  white  matter  of  the  brain  is  a  little  higher  than  in  the  spinal 


62       CHEMICAL  COMPOSITION  OF  NERVOUS  TISSUES    [LECT. 

cord.  This  is  the  only  exception  to  the  general  rule,  and  perhaps 
may  be  explained  by  the  high  percentage  of  neuro-keratin  in 
white  matter.* 

The  earliest  to  study  the  nature  of  the  proteids  was 
Petrowsky.f  He  investigated  the  question  previous  to  our 
modern  ideas  concerning  proteids,  and  described  them  as  con- 
sisting of  a  globulin  somewhat  resembling  myosin,  and  an  albu- 
min especially  abundant  in  grey  matter.  Baumstark,j  in  a  more 
recent  research,  speaks  of  the  chief  proteid  matter  in  nervous 
tissue  as  resembling  casein.  There  is  a  certain  amount  of  truth 
in  this,  for  it  is  a  nucleo-proteid.  A  few  years  later  I  took  up 
the  matter,  and  the  following  were  my  chief  conclusions.! 

The  proteids  present  are  three  in  number.  They  differ  in 
temperature  of  heat  coagulation,  in  the  readiness  with  which  they 
are  precipitated  by  neutral  salts,  and  by  acetic  acid  ;  one  of  them 
contains  phosphorus  and  is  a  nucleo-proteid,  so  differing  from 
the  other  two,  which  are  globulins.  The  most  important  char- 
acters of  these  proteids  are  the  following  : — 

(a)  This  proteid  is  a  globulin ;  it  may  conveniently  be  termed 
neuro-globulin  «.  It  is  coagulated  by  heat  at  the  low  tempera- 
ture of  47°,  and  is  analogous  to  similar  globulins  which  are 
found  in  all  cellular  structures,  such  as  cell-globulin  of  lymph- 
cells,  para-myosinogen  of  muscle,  hepato-globulin  «  ©f  the  liver, 
and  kidney  globulin.  Indeed,  this  proteid  seems  to  be  as  con- 
stant a  constituent  of  protoplasmic  structures  as  the  nucleo- 
proteids  are. 

It  is  precipitated  by  a  comparatively  small  percentage  of 
such  neutral  salts  as  magnesium  sulphate,.  It  is  not  precipitated 
by  weak  acetic  acid.  It  contains  no  phosphorus  in  its  molecule. 

In  view  of  the  subject  of  hyperpyrexia,  a  pathological 
problem  we  shall  be  considering  later,  I  would  ask  you  to  make 
a  mental  note  of  the  low  coagulation  temperature  of  this  proteid. 

*  The  percentage  of  neuro-keratin  is  in  grey  matter,  0.3  ;  in  white  matter, 
2.2  to  2.9  ;  and  in  nerve,  0.3  to  0.6  per  cent.  (Kiihne  and  Chittenden,  Zeit 
/.  Biol.,  vol.  xxvi.,  p.  291). 

t  Pfliiger's  Archiv,  vol.  vii.,  p.  367. 

\  Zeit.f.physioL  Chem.,  vol.  ix.,  p.  145, 

§  Jour,  of  Phys.,  vol.  xv.,  p.  106. 


vi.]  PROTEIDS  OF  NERVOUS  TISSUES  63 

(&)  This  proteid  is  a  nucleo-proteid.  It  can  be  readily  pre- 
pared from  nervous  tissues  by  making  a  saline  extract,  but 
under  these  circumstances  it  is  mixed  with  the  other  proteids. 
It  may,  however,  be  prepared  in  large  quantities  by  precipitat-, 
ing  an  aqueous  extract  of  brain  by  weak  acetic  acid.  The 
supply  obtainable  from  white  matter  is  small. 

It  is  coagulated  by  heat  at  56  to  60°.  Like  globulins,  it  is 
precipitable  by  saturating  its  solutions  with  neutral  salts ;  but 
more  salt  is  necessary  than  in  the  case  of  neuro-globulin  a. 

It  contains  0.5  per  cent,  of  phosphorus. 

After  subjection  to  gastric  digestion,  an  unsoluble  residue  ©f 
nuclein  remains  behind. 

Dissolved  in  dilute  sodium  carbonate  and  injected  into  the 
vascular  system  of  rabbits,  it  causes,  like  other  nucleo-proteids, 
extensive  intravascular  coagulation. 

(c]  This  proteid  is  a  globulin.  It  may  conveniently  be  called 
neuro-globulin  (3,  and  is  closely  analogous  to  the  hepato-globulin 
/3  of  liver  cells.  It  is  coagulated  by  heat  at  70  to  75°  C.  ;  it  is 
precipitable  by  neutral  salts,  but  requires  complete  saturation 
with  magnesium  sulphate  to  precipitate  it  entirely.  It  is  not 
precipitable  by  weak  acetic  acid  like  the  nucleo-proteid  just 
described,  and  contains  no  phosphorus  in  its  molecule. 

The  only  other  research  with  which  I  am  acquainted  on  the 
proteids  of  nervous  tissues  is  one  by  P.  A.  Levene.*  He  has 
particularly  directed  his  attention  to  the  nucleo-proteid  of  the 
brain,  and  although  he  separated  it  out  from  the  organ  by  a 
method  different  from  that  which  I  employed,  he  was  evidently 
dealing  with  the  same  substance ;  the  amount  of  phosphorus 
being  0.5  per  cent,  which  is  the  same  number  I  obtained.  The 
purine  bases  obtainable  from  cerebro-nucleo-proteid  are  guanine, , 
adenine,  small  quantities  of  xanthine,  but  no  hypoxanthine. 

The  Phosphorised  Fats 

The  best  known  member  of  this  group  is  lecithin,  and  the 
compound  of  lecithin  with  cerebrin  which  is  called  protagon. 
But  there  are  several  lecithins  differing  in  the  kind  of  fatty  acid 

*  Archives  of  Neurology  and  Psy  chop  athology,  1899,  vol.  vii.,  p.  14. 


64       CHEMICAL  COMPOSITION  OF  NER  VO  US  TISS UES    [LECT. 

they  contain,  and  other  substances  of  similar  nature  like 
kephalin  which  has  recently  been  the  subject  of  a  research  by 
Waldemar  Koch.  Koch  suggests  the  name  of  the  Lecithans  for 
the  whole  group,  in  place  of  Phosphatides  introduced  by 
Thudichum.  I  myself  prefer  the  expression  pJwsphorised  fats, 
which,  although  it  is  a  little  longer,  is  quite  free  from  ambiguity. 

It  was  in  the  year  1865  tnat  Liebreich  *  separated  from  the 
brain  the  material  he  termed  protagon ;  he  further  found  that 
when  decomposed  by  baryta-water  it  yields  two  acids — stearic 
acid  and  glycero-phosphoric  acid — and  a  base  called  choline. 

Hoppe-Seyler,  and  Diaconowf  working  under  Hoppe-Seyler's 
direction,  denied  the  existence  of  this  substance  protagon,  and 
considered  that  it  was  a  mere  mechanical  mixture  of  the 
phosphorised  fat  called  lecithin,  with  a  nitrogenous  non- 
phosphorised  substance  called  cerebrin.  Lecithin  yields  the 
same  three  decomposition  products  that  were  obtained  from 
protagon  by  Liebreich.  Diaconow's  elementary  analyses  were, 
however,  far  from  convincing. 

The  subject  in  this  country  was  taken  up  by  Gamgee  and 
Blankenhorn ;  J  and  the  result  of  their  work  has  been  that 
Liebreich's  discovery  has  been  fully  verified.  They  showed  that 
protagon  is  a  perfectly  definite  crystalline  substance  of  constant 
elementary  composition.  They  also  showed  that  even  pro- 
longed treatment  with  alcohol  and  ether  will  not  extract  lecithin 
from  protagon,  as  alleged  by  Diaconow.  When  protagon  is 
digested  with  alkalis,  it  yields  cerebrin  or  cerebrins,  and  the 
decomposition  products  of  lecithin. 

This  work  has  been  confirmed  by  Baumstark,§  Ruppel,||  and 
Kossel  and  Freytag.H 

Protagon  is  prepared  by  digesting  brain  with  alcohol  at  45° 
C.,  the  extract  is  filtered  while  warm,  and  then  cooled  to  o°  C. 
Protagon  crystals  mixed  with  cholesterin  are  thus  deposited. 

*  Liebreich,  Annalen  der  Chem.  u.  Pharm.,  vol.  cxxxiv.,  p.  29. 

t  Diaconow,  Centralbl.f.  d.  med.  Wissensch.,  1868,  p.  97. 

J  Gamgee  and  Blankenhorn,  Jour,  of  Phy s.,  vol.  ii.,  p.  113. 

§  Baumstark,  Zeit.  f.  phy siol.  Chem.,  vol.  ix.,  p.  329. 

||  Zeit.f.  Biol.,  vol.  xxxi.,  p.  86. 

1F  Zeit.  f.  phy  siol.  Chem.,  vol.  xvii.,  p.  431. 


vi.]  PROTAGON  AND  LECITHIN  65 

The  cholesterin  is  dissolved  out  by  ether.  The  protagon  is  then 
collected,  redissolved  in  warm  alcohol,  and  allowed  to  recrystal- 
lise  on  cooling.  Its  empirical  formula  was  calculated  by 
Gamgee  and  Blankenhorn  from  their  analytical  results  to  be 
C160H308N5PO35.  But  this  formula  will  require  revision,  as  both 
Kossel  and  Ruppel  have  shown  that  the  molecule  contains  a 
small  amount  of  sulphur  which  had  been  overlooked  by  the 
earlier  observers. 

The  doubt  as  to  the  chemical  individuality  of  protagon  has 
arisen  again  in  recent  years.  Kossel , and  Freytag  in  their  work 
assumed  the  existence  of  several  protagons,  differing  in  the 
nature  of  the  lecithin,  and  also  of  the  cerebrin  they  contain. 
Lesem  and  Gies  *  doubt  whether  protagon  is  a  chemical  unit,  but 
regard  it  as  a  mixture. 

This  is  a  view  which  I  cannot  consider  they  have  proved  ; 
protagon  is  a  definite  crystalline  material ;  it  has  been  analysed 
with  remarkably  concordant  results  by  numerous  investigators. 
Moreover,  Cramer  •(•  has  within  the  last  few  months  prepared 
protagon  by  a  slightly  different  method,  and  shows  that  its 
composition  is  constant.  Allowing  for  the  sulphur,  he  calculates 
its  empirical  formula  to  be  C320H616N10P2SO68.  Its  molecular 
weight  would  therefore  be  5778. 

Although  I  differ  from  Lesem  and  Gies  in  one  of  their 
conclusions,  I  agree  with  another  which  they  put  forward, 
namely,  that  protagon  (whether  it  is  a  unit  or  not),  does  not 
contain  the  bulk  of  the  phosphorised  organic  substances  of 
nervous  material.  There  is  no  doubt  that  the  bulk  of  the  phos- 
phorus is  contained  within  materials  of  smaller  molecular  weight, 
such  as  lecithin  and  kephalin.  The  amount  of  organic  phos- 
phorus is  so  great  that  it  would  be  quite  impossible  for  much  of 
it  to  be  contained  within  a  substance  of  such  high  molecular 
weight  as  protagon. 

Lecithin 

This  may  be  taken  as  a  type  of  the  phosphorised  fats  ;  it  is 
certainly  an  abundant  constituent  of  nervous  tissues,  and  the 

*  American  Jour,  of  Phys.,  1902,  vol.  viii.,  p.  183. 
t  Jour,  of  Phys.,  1904,  vol.  xxxi.,  p.  30. 

E 


66       CHEMICAL  COMPOSITION  OF  NERVOUS  TISSUES   [LECT. 

one  concerning  which  we  have  most  knowledge.  It  is  also  a 
constituent  of  the  protoplasm  of  all  cells.  When  it  is  decom- 
posed, either  in  the  laboratory  or  in  the  body,  it  breaks  up  into 
three  substances,  as  shown  in  the  following  equation : — 

C44H90NP09  +  3H20  =  2C18H3602  +  C3H9P06  +  C5H15NO2 

[lecithin]  [stearic  acid]  [glycero-  [choline] 

phosphoric  acid] 

To  give  this  substance  its  chemical  name,  we  must  call  it 
choline-distearyl-glycero-phosphoric  acid.  The  choline  radicle 
is  united  to  the  acid  by  means  of  the  oxygen  of  the  hydroxyl ; 
it  is  therefore  not  a  salt  but  an  ether-like  combination,  thus  : — 

CH20-C17H36CO 
CHO  -  C17H35CO 

dn2o  -  PO  -  o .  c2H^ 

I  (CH3)3lN. 

OH        HO     J 

The  same  facts  can  be  put  more  simply  by  comparing  an 
ordinary  fat  with  lecithin.  An  ordinary  neutral  fat  such  as 
those  which  are  found  in  adipose  tissue  or  milk  contains  only 
three  elements — carbon,  oxygen,  and  hydrogen.  Lecithin  con- 
tains the  same  three  elements  with  nitrogen  and  phosphorus  in 
addition.  An  ordinary  neutral  fat  on  decomposition  links  to 
itself  the  elements  of  water,  and  then  splits  up  (is  hydrolysed) 
into  glycerin  and  a  fatty  acid  ;  thus  stearin  yields  stearic  acid 
and  glycerin ;  palmitin,  palmitic  acid  and  glycerin  ;  and  olein, 

oleic  acid  and  glycerin* 

Fat  +  water 

Fatty  acid     Glycerin. 

Lecithin  yields  not  only  a  fatty  acid  and  glycerin,  but  in 
addition  to  these  substances  it  gives  rise  ^to  phosphoric  acid, 
which  contains  all  the  phosphorus  of  the  lecithin,  and  choline,  an 
alkaloid  which  contains  all  the  nitrogen  of  the  lecithin. 

Lecithin  +  water 

r        nr          T          ~T. 

Glycerin          Fatty  acid          Phosphoric  acid          Choline. 

The  fatty  acid  in  the  example  we  have  taken  of  a  lecithin 
is  stearic  acid  ;  but,  as  already  stated,  the  acid  obtained  is  not 


VI.]  CHOL1NZ  67 

always  stearic      One  molecule,  at  least,  as  Thudichum  pointed 
out,  is  usually  oleic   acid.     Oleic   acid   is   an   example   of  an 
unsaturated  fatty  acid,  and  hence  its  affinity  for  oxygen.     It 
is  well  known  that  fat  blackens  osmic  acid  (osmium  tetroxide)  ;  - 
that  is,  it  takes  oxygen  from  it  and  forms  a  lower  oxide  having  . 
a  black  colour.     Fats  which  are  saturated,  like  the  glycerides 
of  stearic  and  palmitic  acids,  do  not  give  this  reaction.     The 
black  coloration  which  nerve  fibres  give  with  this  reagent  is 
due  to  the  oleic  acid  they  contain  ;  purified  lecithin  prepared 
from  brain  gives  it  also. 

The  nitrogenous  derivative  of  lecithin,  choline,  is  of  con- 
siderable importance,  because  it  can  be  readily  detected,  and 
the  presence  of  choline  may  be  usually  taken  as  evidence  that 
lecithin,  and  so  nervous  material  generally,  has  undergone 
decomposition.  Choline  is  an  ammonium  base,  which  has  the 
following  constitution. 

f(CH3)3 
N 4  CH2  -  CH2OH 

(OH 

Its  name  was   originally  given  to  it  because  it  was   first 
separated  out  from  the  lecithin  of  the  bile ;  but   its   chemical 
name  is  trimethyl-oxyethyl-ammonium  hydroxide.     It  was  alT>-v 
one  time  thought  to  fre  identical  with  the  base  neurine  which      \ 
Liebreich   separated   from    nervous   tissues,  and   the   two   are 
closely  related,  choline  readily  becoming  converted  into  neurine 
under  the  influence  of  certain  bacterial  agencies.     Neurine  has       / 
also  been  obtained  by  Brieger  from  the  putrefactive  decomposi- 
tion of  flesh.     Neurine  only  differs  from  choline  by  two  atoms 
of  hydrogen  and  one  of  oxygen  ;  and  its  structure, 

HO  .  N  .  (CH3)3C2H3 


is 


justifies  its  chemical  name,  which  is  trimethyl-vinyl-ammonium 
hydroxide.  These  two  alkaloids  are  also  closely  related  to  two 
others,  namely,  betaine,  the  very  slightly  toxic  alkaloid  of  the 
common  beet,  and  muscarine,  the  highly  poisonous  alkaloid  of 
the  toad-stool,  Agaricus  muscarius. 

But  the  lecithins  are  not  the  only  phosphorised  fats  of  the 


68       CHEMICAL  COMPOSITION  OF  NERVOUS  TISSUES    [LECT. 

brain.  An  elaborate  research  by  Thudichum  *  led  him  to  the 
conclusion  that  there  are  three  groups  of  phosphorised  fats  in 
the  brain,  which  he  termed  kephalins,  myelins,  and  lecithins. 

The  kephalins  are  very  soluble  in  ether,  and  do  not  form 
definite  compounds  with  platinum  or  cadmium  as  lecithin  does. 

The  lecithins  are  characterised  by  extreme  instability. 

The  myelins  are  less  soluble  in  ether  than  the  kephalins, 
and  less  soluble  in  alcohol  than  the  lecithins.  They  are  also 
the  most  stable  of  the  phosphorised  fats. 

In  each  of  these  rather  ill-defined  groups  there  are  several 
members,  the  empirical  formulae  of  which  were  calculated. 
Thus  among  the  myelins,  myelin,  paramyelin,  and  amido- 
myelin  were  separated  by  their  varying  solubility  in  different 
reagents.  It  would,  however,  serve  no  useful  purpose  to  go 
more  fully  into  this  matter ;  we  know  little  of  these  substances 
from  the  chemical  standpoint,  and  still  less  from  the  physio- 
logical. 

Waldemar  Kochf  has  recently  repeated  some  of  this  work, 
and  arrives  at  the  following  conclusions  : — 

The  phosphorised  fats  are  found  not  only  in  nervous  tissues^ 
but  in  other  forms  of  protoplasm  ;  for  instance,  he  separated 
lecithin  and  kephalin  from  yeast  cells,  as  well  as  from  brain. 
They  may  therefore  be  fairly  regarded  as  important  for  cell  life. 

Thudichum's  myelins  he  found  in  the  brain  in  small 
quantities  only,  and  did  not  further  investigate  them.  He, 
however,  devoted  a  considerable  share  of  his  work  to  kephalin, 
and  we  may  therefore  briefly  summarise  what  is  known  of  this 
material. 

Kephalin 

Kephalin  may  be  prepared  by  extracting  brain  substance 
with  acetone,  and  subsequently  with  ether.  To  the  extracts 
alcohol  is  added  which  precipitates  the  kephalin.  This  pre- 
cipitate is  collected,  dissolved  in  ether,  and  purified  by  repeated 

*  Report  of  Med.  Officer  of  the  Privy  Council,  1874,  p.  113  ;  and  A 
Treatise  on  the  Chemical  Constitution  of  the  Brain,  1884. 

t  The  Lecithans,  The  Decennial  Publications  of  Chicago  University, 
1902,  vol.  x. ;  American  Jour,  of  Phys.,  1904,  vol.  xi.,  p.  303. 


vi.j  KEPHALIN  AND  CEREBRINS  69 

solution  and  reprecipitation.  Thudichum  ascribed  to  this  princi- 
pal member  of  his  kephalin  group,  the  formula  C42H79NPO13, 
and  among  its  decomposition  products  identified  choline,  and 
a  fatty  acid  he  termed  kephalic  acid.  He  considered  that 
kephalic  acid  took  the  place  of  the  oleic  acid  of  lecithin. 

Koch's  analytical  figures  differ  somewhat,  though  not  very 
greatly,  from  Thudichum's : — 

C        H  N  P 

Thudichum    .        .     60        9.38         1.68        4.27  per  cent. 
W.  Koch        .        .     59.5      9.7          1.78        3.84    „      „ 

The  most  important  difference  is  in  the  phosphorus,  which, 
according  to  Koch,  is  present  in  the  same  amount  as  in  lecithin. 

All  we  can  at  present  say  about  kephalin  is  that  it  is  a 
member  of  the  group  of  phosphorised  fats,  and  it  is  possible  as 
Koch  suggests  that  it  may  be  a  stage  in  lecithin  metabolism. 
Whether  kephalic  acid  is  a  new  fatty  acid,  or  merely  oleic 
acid  contaminated  with  other  fatty  acids,  it  is  impossible  to 
say. 

Miss  Tebb  has  recently  in  my  laboratory  prepared,  from 
brain  considerable  quantities  of  lecithin  and  kephalin,  with  a 
view  to  settling  some  of  these  vexed  points  if  possible, 
Kephalin  gives  the  osmic  acid  reaction  in  a  very  intense  way  ;  - 
so  if  its  fatty  acid  is  not  oleic,  it  is  probably  a  member  of  the 
same  series. 

The  Cerebrins 

These  are  nitrogenous  substances  which  are  found  in  the 
white  substance  (especially  in  the  medullary  sheaths),  and  also 
in  egg  yolk,  pus  corpuscles,  spleen  cells,  etc.  Several  members 
of  the  group  have  been  separated  and  analysed  since  they  were 
first  described  by  Miiller.*  Muller's  formula  for  cerebrin  is 
C34H33NO6.  Thudichum's  phrenosin  (C34HNO8),  and  kerasin 
(C46H91NO9)  fall  into  the  same  group,  as  does  also  Gamgee's 
pseudo-cerebrin  (C44H92NO8).  At  present,  however,  these 
materials  have  not  advanced  into  the  area  of  practical  medicine. 
Suffice  it  to  say  that  the  other  name  of  cerebroside  given  to  the 
group  indicates  that  they  are  glucosides,  and  that  the  sugar 
*  Annal,  d.  Chem.  u.  Pharm^  vol.  cv.,  p.  361,  2nd  Abth. 


70       CHEMICAL  COMPOSITION  OF  NER  VO  US  TISS  UES    [LECT. 

(cerebrose)  obtainable  from  them  has  been  identified  as 
Pfflactose  almost  simultaneously  in  this  country  and  in 
Germany.* 

Gamgee  t  was  the  earliest  to  contest  Hoppe-Seyler's  view  that  protagon 
is  a  mere  mixture  which  can  be  separated  by  the  action  of  solvents  into  a 
phosphorised  substance,  and  a  non-phosphorised  cerebrin.  He,  however, 
admitted  that  by  more  vigorous  means  (action  of  caustic  baryta)  cerebrins 
are  obtainable  from  protagon  ;  of  which  in  particular  he  studied  the  one  he 
named  pseudo-cerebrin.  Recently  Woerner  and  Thierfelder  \  have  isolated 
a  substance  they  called  cerebron,  by  treating  protagon  with  alcohol  contain- 
ing benzene.  Cramer  points  out  that  this  is  probably  pseudo-cerebrin 
under  another  name  ;  their  analytical  figures  certainly  closely  agree  with 
those  of  Gamgee. 

THE  CEREBRO-SPINAL  FLUID 

Any  account  of  the  chemical  structure  of  the  brain  would  be 
incomplete  without  some  reference  to  the  cerebro-spinal  fluid, 
and  I  propose  to  conclude  this  lecture  by  a  brief  description  of 
this  remarkable  fluid. 

Most  of  our  knowledge  of  the  cerebro-spinal  fluid  has  been 
derived  from  an  examination  of  the  contents  of  meningoceles, 
and  in  cases  of  hydrocephalus.  The  fluid  removed  by  the  first 
tapping  at  all  events  may  be  regarded  as  fairly  normal.  A  few 
years  ago,  however,  I  had  the  opportunity  of  examining  the 
fluid  from  a  remarkable  case  which  was  under  the  care  of  Dr 
St  Clair  Thomson.  The  patient  was  a  young  woman  who  had 
for  years  suffered  from  continuous  dripping  from  the  nose  ;  this 
was  not  amenable  to  any  treatment.  At  first  it  was  thought  to 
be  a  case  of  nasal  hydrorrhcea,  but  certain  characters  in  the 
affection  convinced  the  observer  that  this  could  not  be  so,  and 
that  the  fluid,  which  dropped  from  one  nostril  only,  was  cerebro- 
spinal  fluid.  This  was  supported  by  the  results  of  the  chemical 
examination  of  the  fluid. 

The  escape  of  cerebro-spinal  fluid  from  the  nose  has  long 
been  known  to  follow  traumatic  injury  to  the  cribriform  plate 

*  Brown  and  Morris,  Proc.  Chem.  Soc.,  London,  1889,  p.  167  ;  Thier- 
felder Zeit  f.  physiol.  Chem.,  vol.  xiv.,  p.  209. 
t  Phys.  Chem.,  vol.  i.,  p.  440  et  seq. 
\  Zeit.  f.  physiol.  Chem.,  1900,  vol.  xxx.,  p.  542. 


vi.]  THE  CEREBRO-SP1NAL  FLUID  71 

of  the  ethmoid  bone,  but  the  possibility  of  its  spontaneous 
escape  from  the  nose  does  not  appear  to  have  been  fully 
established  before  the  present  instance.  However,  considerable 
research  into  the  literature  of  the  subject  has  shown  that  there 
are  several  cases  recorded  in  which,  though  no  history  of  injury 
existed,  the  flow  of  fluid  from  the  nose  was  of  such  a  character 
that  they  must  have  been  similar  to  the  present  case,  although 
in  the  majority  of  instances  the  true  nature  of  the  fluid  escaped 
observation.  Many  of  these  patients  exhibited  cerebral  symp- 
toms in  the  course  of  the  disease,  and  some  ultimately  died  from 
inflammation  of  the  cerebral  meninges,  which  had  probably 
spread  from  the  nose  through  some  opening  in  the  bony  lamina 
that  normally  separates  the  cranial  and  nasal  cavities.* 

The  first  inquiries  we  instituted  in  Dr  Thomson's  case 
related  to  the  quantity  of  fluid  formed.  One  portion,  collected 
by  the  patient  herself  in  the  course  of  an  hour,  measured  4  c.c. 
Another  portion,  collected  under  the  supervision  of  Dr  Thomson 
in  ten  minutes,  measured  3.9  c.c. 

If  the  first  portion  is  taken  as  a  measure  of  the  rate  of 
secretion,  the  amount  formed  in  the  day  will  be  96  c.c.  Taking, 
however,  the  second  observation  as  being  more  accurate,  the 
amount  formed  in  the  twenty-four  hours  will  be  over  half  a  litre 
(561.6  c.c.).  It  is  possible  that  this  estimate  is  too  high,  as 
doubtless  the  patient,  being  under  the  observation  of  a  physician, 
would  be  somewhat  excited,  and  the  consequent  alteration  of 
the  circulation  would,  as  we  shall  immediately  see,  cause  the 
flow  to  become  more  abundant. 

In  a  monograph  on  the  cerebral  circulation  f  Hill  put  forward 
the  view  that  the  rate  of  secretion  of  the  cerebro-spinal  fluid, 
when  the  cranio-vertebral  cavity  is  opened,  depends  directly  on 
the  difference  between  the  pressure  in  the  cerebral  capillaries 
and  that  of  the  atmosphere.  At  the  same  time  it  was  shown  that 

*  "  Observations  on  the  Cerebro-spinal  Fluid  in  the  Human  Subject,"  by 
St  Clair  Thomson,  M.D.,  Leonard  Hill,  M.B.,  F.R.S.,and  W.  D.  Halliburton, 
M.D.,  F.R.S.,  Proc.  Roy.  Soc.,  vol.  Ixiv.,  p.  343. 

A  full  account  of  the  case  is  given  in  Dr  Thomson's  book,  The  Cerebro- 
spinal  Fluid.  Cassell  &  Co.,  1899. 

t  The  Physiology  and  Pathology  of  the  Cerebral  Circulation^  by  Leonard 
Jiill.  London  :  Messrs  Churchill,  1896. 


72       CHEMICAL  COMPOSITION  OF  NERVOUS  TISSUES    [LECI. 

cerebral  capillary  pressure  varies  directly  and  absolutely  with 
-  vena  cava  pressure.  Thus  the  cerebral  capillary  pressure  can  be 
raised  with  great  ease  by  any  agency  which  causes  a  rise  of 
pressure  in  the  vena  cava  or  cerebral  veins.  On  the  other  hand, 
cerebral  capillary  pressure  varies  directly,  but  only  proportion- 
ately, with  aortic  pressure,  for  between  the  aorta  and  the  capil- 
laries there  lies  the  peripheral  resistance. 

It  follows  from  the  above  that  the  easiest  methods  of  raising 
the  cerebral  capillary  pressure  in  man  are :  (a)  By  compression 
of  the  abdomen,  (fr)  By  the  assumption  of  the  horizontal 
posture.  In  this  position,  however,  the  rise  of  venous  pressure 
may  be  compensated  by  the  fall  of  arterial  pressure,  which 
normally  occurs  when  the  body  is  at  rest ;  this  is,  no  doubt, 
the  case  during  sleep.  (<:)  By  straining  or  forced  expiratory 
effort,  with  the  glottis  closed. 

By  all  these  methods  the  vena  cava  pressure  is  considerably 
raised  ;  and  by  the  last  method  the  venous  inlets  into  the  thorax 
may  be  completely  blocked,  and  the  pressure  in  the  cerebral 
capillaries  raised  to  something  like  aortic  pressure. 

It  is  true  that  by  such  a  forced  expiratory  effort  the  aortic 
pressure  is  lowered.  Nevertheless,  the  total  effect  on  capillary 
pressure  is  a  very  great  rise,  for  a  fall  of  aortic  pressure  of  25  mm. 
of  mercury  produces  a  fall  in  cerebral  capillary  pressure  of  less 
than  5  mm.  of  mercury,  while  a  rise  of  vena  cava  pressure  of 
25  mm.  of  mercury  produces  a  rise  of  cerebral  capillary  pressure 
of  25  mm.  Hg. 

Dr  Thomson's  case  gave  us  a  unique  opportunity  of  testing  the 
correctness  of  these  views  on  the  living  human  subject,  and  our 
experiments  entirely  confirm  them.  As  will  be  seen  from  the 
following  figures,  the  flow  of  cerebro-spinal  fluid  is  accelerated  by 
all  those  circumstances  which  raise  the  cerebral  capillary  pressure. 

The  fluid  passed  while  the  patient  was  making  forced  expira- 
tory efforts  was  nearly  double  in  quantity  that  which  flowed 
while  she  was  sitting  quietly.  Abdominal  compression  also 
raised  the  rate  of  flow,  by  increasing  the  vena  cava  pressure  and 
so  leading  to  increase  of  the  cerebral  capillary  pressure.  In  all 
cases  increase  of  volume  is  accompanied  with  fall  in  the  percent- 
age of  solids  in  the  fluid, 


vi.]  THE  CEREBRO-SPINAL  FLUID 

The  following  table  illustrates  these  points  :— 


73 


Condition  of  Patient. 

Amount  of  fluid 
collected  in 
ten  minutes. 

Percentage  of  solids 
in  the  fluid. 

I.  Sitting  quietly  .... 
2.  During  straining  .... 

2.378  c.c. 
3.912  c.c. 

I.I 

0.43 

I.  Sitting  quietly  .... 
2.  Abdomen  compressed 

2.188  C.C. 

3.009  c.c. 

1.14 
0.68 

I.  Sitting  upright  .  .  ".  . 
2.  Lying  down  ..... 

1.670  c.c. 
3-245  c.c. 

i.  ii 
1.03 

Cavazzani,*  from  experiments  on  dogs,  found  that  the  cerebro- 
spinal  fluid  collected  in  the  morning  was  more  alkaline  than  in 
the  evening,  and  contained  more  solid  residue.  He  considers 
that  this  is  related  to  the  activity  of  the  nervous  system,  and  that 
it  confirms  Obersteiner's  theory -of  sleep.  He  obtained  corre- 
sponding results  in  the  case  of  a  man  with  traumatic  fistula  of 
the  frontal  bone. 

We  considered  it  worth  while  to  repeat  this  observation. 

The  qualitative  examination  of  the  fluid  collected  first  thing 
on  several  mornings  gave  the  same  results  as  that  of  specimens 
collected  the  last  thing  in  the  evening.  Both  were  distinctly 
alkaline  to  litmus,  but  no  estimation  of  the  relative  alkalinity  - 
was  made.  The  following  table  gives  in  percentages  the  results 
of  the  quantitative  analyses  : — 


Morning  Fluid. 

Evening  Fluid. 

Water.         . 
Solids  .         .         ..'... 
Organic  solids 
Inorganic  solids      .... 

99.004 
0.996 
0.118 
0.878 

99.027 
0.973 
O.IOO 

0.873 

The  evening  fluid  is  thus  slightly  poorer  in  both  classes  of 
constituents  than  that  of  the  morning ;  the  difference  is  chiefly 

*  "  Sul  Liquido  Cerebrospinale,"  La  Riforma  Medica^  Anno  VIII.,  1892, 
vol.  ii.,  p.  591. 


74       CHEMICAL  COMPOSITION  OF  NER  VO  US  TISS  UES    [LECT. 

due  to  an  alteration  in  the  organic  solids.  This  is  just  what  we 
should  expect,  because  the  decreased  capillary  pressure  during 
sleep  would  lessen  the  rate  of  exudation  of  water.  Without  com- 
mitting ourselves  to  any  theory  on  nervous  activity  or  sleep,  we 
may  say  that  our  experiments  confirm  those  of  Cavazzani, 

All  our  experiments,  therefore,  show  the  close  correspondence 
between  the  amount  of  the  fluid  and  the  height  of  cerebral 
capillary  pressure.  But  in  spite  of  this,  cerebro-spinal  fluid  is 
not  a  simple  pressure  exudation  from  the  blood,  The  idea  that 
it  is  a  secretion  was  first  propounded  by  Carl  Schmidt  many 
years  ago,  long  before  the  birth  of  Heidenhain's  theory  that  all 
lymph  must  be  regarded  as  a  secretion,  in  the  formation  of  which 
the  endothelial  cells  of  the  capillaries  play  a  selective  action. 

Schmidt  propounded  his  doctrine  on  the  strength  of  his 
analyses  of  the  saline  constituents  of  the  fluid  ;  he  stated  that 
potassium  salts  are  more  abundant  than  those  of  sodium.  But 
this  has  not  been  confirmed  by  subsequent  investigators.  Thus 
Yvon*  gives  the  following  numbers,  NaCl  7.098,  and  KC1  0.033 
per  1000.  F.  Mullerf  gives  the  relationship  of  NaCl  to  KC1  as 
21.5  :  i.  My  own  figures];  in  cases  of  meningocele  show  in  100 
parts  of  chlorides  that  95.15  consist  of  sodium  chloride,  and  4.85 
of  potassium  chloride. 

The  amount  and  proportions  of  the  salts  are  thus  about  the 
same  as  in  blood,  lymph,  and  transudations  generally. 

But  examination  of  the  organic  solids  shows  Schmidt's  con- 
tention that  cerebro-spinal  fluid  is  a  secretion  to  be  correct, 
though  the  grounds  on  which  he  supported  his  idea  are  in- 
correct. 

The  fluid  stands  apart  from  all  other  similar  fluids  in  the 
following  particulars  : — 

(1)  Its  clear,  watery  character. 

(2)  Its  low  specific  gravity  (1004  to  1007). 

(3)  It  only  contains  a  trace  of  proteid  ;  the  characters  of  this'- 
are  those  of  a  globulin,  whilst  in  some  cases  a  small  admixture 
of  proteose  is  present.     Albumin  and  fibrinogen  are  absent. 

*  Jour,  de  Pharm.  et  de  Chemie,  fourth  series,  1877,  vol.  xxvi.,  p.  240. 
t  Mittheil.  a.  d.  Wiirzburger  med.  Klinik,  vol.  i.,  p.  267. 
\  Jour,  of  Phys.)  vol.  x.,  p.  232. 


VI.] 


THE  CEREBRO-SPINAL  FLUID 


75 


(4)  The  presence  in  it  of  a  substance  which  reduces  Fehling's 
solution. 

For  some  reason  or  other  this  does  not  readily  give  the  fermentation  test 
with  yeast.  Hence  for  many  years  the  statement  was  current  that  it  could 
not  be  sugar.  At  one  time,  from  the  examination  of  a  case  in  which  a  large 
amount  of  fluid  was  at  my  disposal,  I  put  forward  the  hypothesis  that  the 
reducing  substance  was  an  aromatic  body  allied  to  pyrocatechin.  This 
illustrates  the  danger  of  drawing  conclusions  from  an  insufficient  number  of 
observations.  Nawratski,*  however,  with  new  methods  at  his  disposal  for 
identifying  sugars,  was  able  to  definitely  prove  that  in  the  calf,  the  reducing 
substance  is  dextrose.  Since  then,  other  observers  have  had  no  difficulty  in 
substantiating  Nawratski's  statement  in  regard  to  the  fluid  of  other  animals, 
man  included. 

The  following  analyses  (in  parts  per  1000)  of  the  fluid  from 
spina  bifida  cases  may  be  next  given  •(• : — 


In  parts  per  1000. 

Case  1. 

Case  2. 

Case  3. 

Water       

080.71; 

989.877 

991.658 

Solids       

10.25 
0.842 

IO.I23 
1.  602 

8.342 
0.199 

Extractives) 
Salts            j 

9.626 

/         0.631 
I          7.890 

3.028 
5."5 

The  percentage  of  organic  solids  is  thus  as  a  rule  a  little 
higher  than  in  the  absolutely  normal  fluid.  In  cases  of  hydro- 
cephalus  the  percentage  of  solids  is  rather  greater  (see  next 
table). 


In  parts  per  1000. 

Case  1. 

Case  2. 

Case  3. 

Water 

986.78 

084.  t;o 

980.77 

Solids       .         .         .    •     V"      . 

13.22 

15.41 

19.23 

Proteids  and  extractives    . 
Salts          

3-74 
9.48 

6.49 
8.92 

11-35 
7.88 

In  cases  of  chronic  hydrocephalus,  the  fluid  removed  by  the 
first    tapping    has   the    normal    qualitative    characteristics    of 

*  Zeit.f.physiol.  Chem.,  1897,  vol.  xxiii.,  p.  523. 

A  W.  D.  Halliburton,  "  Report  of  Spina  Bifida  Committee,"  vol.  xviii.  of 
Clin.  Soc.  Transactions. 


76       CHEMICAL  COMPOSITION  OF  NERVOUS  TISSUES   [LECT. 

cerebro-spinal  fluid  ;  but  that  removed  by  subsequent  tappings 
resembles  a  dilute  transudation  from  the  blood,  and  if  inflam- 
mation supervenes  this  becomes  more  marked  ;  the  proteids 
become  more  abundant,  and  resemble  those  found  in  blood 
and  lymph ;  the  amount  of  reducing  substance  increases  also. 
This  is  illustrated  in  the  following  table : — 

Case  of  Chronic  Hydrocephalus. 


Specific 
Gravity. 

Percentage  of 
Proteids. 

Reducing  Substance. 

First  tapping 
Second  tapping 
Third  tapping 

1006 
1010 
1010 

0.045 
0.069 
0.272 

Traces. 
Fairly  abundant. 
More  abundant. 

It  is  an  interesting  question  whether  the  fluid  has  the  same 
composition  in  all  parts,  for  the  fluid  has  a  double  origin.  It  is 
found  in  the  lymph  channels  and  spaces  of  the  brain  and  cord 
tissue,  and  the  perivascular  lymphatics  have  been  shown  to 
open  into  the  subarachnoid  space.  In  the  second  place,  it  is- 
found  in  the  cerebro-spinal  cavity  (ventricles  of  brain  and  central 
canal  of  cord),  and  it  can  hardly  be  doubted  that  it  is  here 
formed  largely  by  the  secretory  epithelial  cells  which  cover  the 
choroid  plexuses.*  We  can  only  surmise  that  this  double 
method  of  formation  may  imply  a  difference  in  the  composition 
of  the  fluid  formed.  The  fluid  as  usually  examined  must  be  a 
mixture  of  the  two,  and  I  cannot  see  that  we  have  at  present 
any  certain  method  of  collecting  the  two  fluids  separately. 

I  will  conclude  with  one  more  question,  and  that  is,  whether 
choline  occurs  in  normal  cerebro-spinal  fluid.  The  importance 
of  this  question  arises  from  the  fact  that  choline  is  a  substance 
which  lends  itself  readily  to  detection,  and  its  presence  is  a 
valuable  indication  of  a  breakdown  of  nervous  tissue.  In  a 
fluid  which  plays  the  part  of  a  lymph,  we  naturally  look  for  the 
products  of  disintegration  of  any  tissue.  Mott  and  I  have 
shown  that  in  diseased  conditions  in  which  the  katabolic  side  of 


*  See  article  on  Meningitis,  by  Dr  Lees  and  Sir  T.  Barlow,  in  Allbutt's 
System  of  Medicine. 


vi. j  THE  CEREBRO-SPINAL  FLUID  77 

nervous  action  is  preponderant,  choline  is  found  in  great 
abundance.  This  is  a  point  I  shall  have  to  dwell  upon  more 
fully  later.  For  the  present  it  is  sufficient  to  say  that  in  the 
normal  fluid  so  little  is  present  that  it  may  be  regarded  as 
absent  for  all  practical  purposes.  Still  it  is  present.  This 
Gumprecht  *  has  shown  to  be  the  case ;  he  worked  with  larger 
quantities  of  fluid  than  were  used  by  Mott  and  myself. 
Although  the  quantity  in  the  normal  fluid  is  so  small,  it  is  not 
without  interest,  for  it  furnishes  evidence  that  in  the  metabolism 
of  the  nervous  tissues  lecithin  as  well  as  proteid  is  in  a  condition 
of  unstable  chemical  equilibrium. 

The  osmotic  relationships  of  cerebro-spinal  fluid  are  different  from  thbse 
of  ordinary  lymph. 

Thus  Zanier  t  finds  in  the  ox  that  the  fluid  is  hypertonic  compared  to  the 
serum  of  the  same  animal  Widal,  Sicard,  and  Ravant  \  arrived  at  the 
same  result  by  the  cryoscopic  method.  This  character  separates  it  from 
other  serous  fluids,  and  various  drugs  pass  from  the  cerebro-spinal  fluid  into 
the  blood. 

M.  Lewandowsky  §  has  performed  somewhat  similar  experiments  ;  he 
regards  the  fluid  as  a  specific  product  of  the  brain,  and  only  to  a  small 
extent  as  a  simple  transudation  from  the  blood. 

The  experiments  of  F.  Ransom  ||  with  tetanus  toxin  and  antitoxin  show 
that  these  organic  materials  will  pass  from  the  cerebro-spinal  fluid  to  the 
blood,  though  they  pass  in  the  opposite  direction  from  the  blood  to  the 
lymph. 

*   Verhandl.  des  Congr.  /.  innere  Med.,  Wiesbaden,  1900,  pp.  326-348. 

t  CentrabLf.  Physiol.,  1896,  vol.  x. 

%  La  Presse  medicale^  October  24,  1900,  p.  128. 

§  Zeit.  klin.  Med.,  vol.  xl.,  p.  480. 

1 1  Zeit.  f.  physiol.  Chem.^  1900,  vol.  xxxi. 


LECTURE   VII 

METABOLISM   IN    NERVOUS   TISSUES 

IN  my  last  lecture  I  dwelt  upon  the  general  composition  of 
nervous  structures,  and  in  addition  to  giving  you  a  long  list  of 
the  chemical  substances  found  there,  with  tables  of  quantitative 
analyses,  devoted  some  time  to  a  description  of  the  proteids, 
and  phosphorised  constituents  of  nervous  material. 

I  propose  to  ask  you  to-day  to  follow  me  in  the  inquiry  as 
to  the  evidence  we  possess  of  metabolic  activity  in  nervous 
tissues.  This  will  involve  the  discussion  of  such  questions  as 
fatigue  and  sleep. 

To  ascertain  the  chemical  composition  of  the  brain  when  it 
is  dead,  is  a  task  of  some  difficulty,  but  it  is  easy  when  compared 
with  the  endeavour  to  determine  what  chemical  changes  it 
undergoes  while  it  is  alive.  We  are  only  on  the  threshold  of 
such  chemical  inquiries ;  still  the  time  cannot  be  far  distant 
when  we  shall  have  crossed  it  and  opened  the  door  to  more 
certain  knowledge.  We  shall  find  here  that  the  experiments 
made  for  us  by  nature,  which  we  call  diseases,  will  come  to  our 
aid,  for  in  pathological  conditions  we  have  not  the  nicely 
balanced  equilibrium  between  anabolism  and  katabolism  which 
characterises  the  physiological  state,  but  as  a  rule  the  katabolic 
side  predominates,  and  so  we  are  enabled  to  grasp  it. 

Very  often,  for  the  purposes  of  teaching  and  illustration,  we 
compare  the  nervous  system  to  a  telegraphic  system,  penetrating 
to  every  part  of  a  country  and  serving  for  the  regulation  and 
ordering  of  the  various  occurrences  which  take  place  there. 
Messages  fly  to  and  from  distant  parts,  and  are  received,  co- 
ordinated or  started  at  central  offices,  which  we  may  compare 
to  the  groups  of  nerve-cells  we  call  nerve-centres.  In  such  a 

78 


LECT.  VIL]      METABOLISM  IN  NERVOUS  TISSUES  79 

telegraphic  system,  the  most  active  parts  are  the  offices  ;  it  is 
there  we  look  for  evidence  of  action  in  the  shape  of  fatigue  in 
the  operators,  or  wear  and  tear  of  instruments.  The  wires  are, 
comparatively  speaking,  passive  transmitters  ;  they  undergo  but 
little  change,  and  do  not  manifest  signs  of  fatigue. 

So  it  is  in  the  nervous  system  ;  the  signs  of  action  are  to  be 
found  in  the  beginnings  and  endings  of  the  nerve-fibres,  the 
cells  of  brain  and  cord,  and  the  end  organs  in  muscle  and  other 
peripheral  structures.  Any  evidence  of  fatigue  in  the  more 
passive  transmitters,  the  nerve-fibres,  is  very  difficult  to  discover. 
This  coincides  with  the  arrangements  of  the  vascular  supply  of 
such  parts.  The  nerve-centres  are  richly  supplied  with  blood- 
vessels, which  furnish  them  with  an  abundant  supply  of  nutrient 
material.  Cerebral  anaemia  rapidly  produces  pathological 
changes  in  the  nerve-cells,  and  death  quickly  supervenes.  But 
in  a  nerve  the  blood-vessels  are  comparatively  insignificant,  and 
a  nerve  can  be  removed  from  the  body,  and  be  made  to  manifest 
activity  for  many  hours  subsequently ;  though,  even  here,  as 
Verworn,  Baeyer,  and  Frohlich  have  shown,  oxygen  is  essential. 
The  question  arises  here,  as  in  muscle,  whether  the  oxygen  is 
more  important  for  the  anabolic  or  the  katabolic  side  of  nervous 
metabolisms. 

The  necessity  for  oxygen,  and  the  fact  that  it  is  used  up 
during  the  activity  of  the  brain,  can  be  very  strikingly 
demonstrated  by  an  experiment  which  Hill  performed  with 
the  help  of  methylene  blue.  Ehrlich  was  the  first  to  show 
that  if  a  solution  of  this  pigment  is  injected  into  the  blood 
stream  of  an  animal,  the  blood  is  rendered  blue,  but  the  organs, 
especially  those  which  like  glandular  organs  are  in  a  state  of 
activity,  are  colourless.  On  exposure  to  oxygen  after  the  organs 
are  removed  from  the  body,  they  also  become  blue.  The 
meaning  of  this  is,  the  seat  of  oxidation  is  in  the  tissues  and  - 
not  in  the  blood.  Though  methylene  blue  holds  its  oxygen 
more  firmly  than  oxyhaemoglobin  does,  the  tissues  are  never- 
theless able  to  take  oxygen  from  it  and  form  a  colourless 
reduction  product ;  but  after  the  tissues  are  removed  from  the 
body,  and  consequently  are  losing  this  vital  avidity  for  oxygen, 
they  become  blue  once  more  on  exposure  to  the  atmosphere. 


8o  METABOLISM  IN  NERVOUS  TISSUES  [LECT. 

Now,  in  an  anaesthetised  animal  the  brain  is  inactive,  and 
the  brain,  like  the  blood,  has  a  blue  tint.  If,  however,  a  spot  of 
the  cerebral  surface  is  stimulated,  that  part  of  the  brain  is 
thrown  into  action,  oxygen  is  used  up,  and  the  methylene  blue 
is  reduced,  and  in  consequence  that  area  of  the  brain  loses  its 
blue  colour.  If  the  animal  is  so  deeply  narcotised  that  the 
brain  does  not  discharge  an  impulse,  the  part  stimulated 
remains  blue. 

In  any  plan  of  research  on  changes  in  nerve,  we  must  be 
largely  guided  by  what  is  already  known  of  the  tissue  to  which 
it  is  so  closely  related,  namely,  muscle. 

When  a  muscle  is  active,  the  changes  it  undergoes  are 
numerous  and  easy  to  detect.  The  naked  eye  can  see  its 
shortening ;  the  microscope  reveals  changes  in  its  constituent 
sarcous  elements.  The  production  of  heat  is  so  prominent  that 
a  temporary  rise  of  temperature  can  be  ascertained  to  occur 
even  with  such  a  rough  instrument  as  a  thermometer,  though 
for  the  finer  changes  in  the  temperature  of  small  muscles  a 
thermopile  is  necessary.  Accompanying  these  manifestations 
of  a  transformation  of  energy,  the  galvanometer  shows  us  an 
electrical  variation ;  and  the  basis  of  all  the  other  changes  is 
the  sudden  and  massive  increase  of  its  normal  chemical  tone. 

Turning  to  nervous  tissues,  what  a  contrast  we  have. 
When  active,  no  change  is  visible  to  the  highest  powers  of  the 
microscope ;  the  refractive  index  of  the  axis  cylinder  remains 
unaltered;*  the  most  delicate  thermopile  fails  to  detect  any 
rise  of  temperature,  and  the  chemical  changes  which  occur  are 
proved  to  take  place  rather  by  circumstantial  than  by  direct 
evidence.  The  only  change  in  an  isolated  nerve  which  can  be 
detected  by  physical  means  is  the  electrical  variation. 

The  chemical  changes  that  occur  on  the  death  of  a  muscle 
are  in  part  an  exaggeration  of  those  which  take  place  when  it  is 
active  during  life.  This  is  a  guide  to  us  when  we  seek  to 
determine  the  corresponding  facts  in  nerve.  Rolleston  f  showed 
that  in  nerve  there  is  on  its  death  a  rise  of  temperature.  Now 
this  can  only  be  due  to  increased  chemical  action,  and  probably 

*  Grose,  Pfliiger's  Archiv,  vol.  xlvi.,  p.  56. 
+  Jour,  of  Phys.,  vol.  xi.,  p.  208. 


vii.]  REACTION  OF  NERVOUS  TISSUES  81 

of  the  same  kind  as,  though  greater  in  degree  than,  that  which 
occurs  during  life.  Moreover,  nervous  tissues  become  acid  when 
they  die. 

But  in  order  to  systematise  the  description  of  these  changes, 
it  will  be  best  to  consider  them  under  the  following  headings  : — 

(1)  The  reaction  of  nervous  tissue. 

(2)  The  hypothetical  production  of  carbonic  dioxide  during 
the  activity  of  nerve. 

(3)  Evidence    of   metabolic   activity    in    nervous   structures 
derived  from  the  examination  of  cerebro-spinal  fluid,  and  saline 
extracts  of  nervous  tissues. 

(4)  Evidence  of  metabolic  activity  in  nervous  centres  derived 
from  histological  examination  of  nerve-cells. 

(5)  The  absence  of  fatigue  changes  in  nerve-fibres. 

(6)  Sleep  and  narcosis. 

Reaction  of  Nervous  Tissues 

Heidenhain  *  and  Gschleidlen  f  both  state  that  the  normal 
reaction  of  the  axis-cylinder  is  alkaline ;  but  on  death  or  on 
long-continued  activity  the  reaction  becomes  acid.  They 
further  state  that  the  grey  matter  is  acid  even  during  life.  O. 
Langendorffj  found  the  reaction  of  the  central  nervous  system 
alkaline  during  life ;  the  alkalinity  rapidly  diminishes  after 
death,  or  on  stoppage  of  the  circulation.  S.  Moleschott  and 
Battistini  §  found  both  central  and  peripheral  portions  of  the 
nervous  system  acid,  especially  the  grey  matter ;  this  was 
increased  by  activity. 

I  am  convinced  that  these  conflicting  statements  are  in  part 
due  to  the  fact  that  the  nervous  structures  in  question  were  not 
always  examined  in  the  perfectly  fresh  condition,  and  they  may 
also  be  partly  explained  by  the  use  of  different  indicators  of 
acidity  by  the  various  observers. 

In   my  own  work,  I  have  found  in  animals  that  the  fresh 

*  Centralblf.  d.  med.  Wiss.,  1868,  p.  833. 
t  Pftxiger's  Archiv,  vol.  viii.,  p.  171. 

|  Neurol.  CentralbL,  1885,  No.  14.     CentralbL  f.  d.  med.  Wiss.,  1886,  No. 
25.     See  also  Miiller  and  Ott,  Pfliiger's  Archiv,  1904,  vol.  ciii.,  p.  493. 
§  Arch.  ital.  de  biol.,  vol.  viii.,  p.  90.     Chem.  Centr.-BL,  1887,  p.  1224. 

F 


82  METABOLISM  IN  NERVOUS  TISSUES  [LECT. 

tissues  are  invariably  alkaline,  but  on  exposure  they  become 
rapidly  acid,  especially  the  grey  matter.  In  the  human  brains  I 
received  from  the  post-mortem  room  the  reaction  of  the  grey 
matter  was  always,  and  of  the  white  matter  often,  acid  to  litmus. 
This  I  attribute  to  changes  after  death,  for  at  least  twenty-four 
hours  had  always  elapsed  since  death.  The  acidity  is  due  to 
lactic  acid ;  but  according  to  Muller  and  Gscheidlen  it  is  not 
sarcolactic  acid  but  the  fermentation  lactic  acid.  Muller  also 
obtained  traces  of  fonnic  acid. 

In  order  to  test  the  question  of  whether  acidity  develops  on 
activity,  Brodie  and  I  investigated  what  occurs  in  a  non- 
medullated  nerve.  This  appeared  to  us  the  best  means  of 
attacking  the  problem,  for  the  possibly  masking  effect  of  a  large 
mass  of  myelin  would  then  be  absent.  The  splenic  nerves  of 
the  dog,  which  are  large  and  easily  dissected  out,  were  used,  but 
we  found  that  after  faradisation  for  six  hours  the  reaction  never 
became  acid  to  litmus. 


The  Hypothetical  Production  of  Carbon  Dioxide  during  the 
Activity  of  Nerve 

This  is  an  interesting  branch  of  the  subject,  which  we  owe 
to  Dr  Waller.  Waller  uses  as  his  object  of  attack  isolated 
nerves,  usually  the  sciatic  nerves  of  frogs.  He  stimulates  them 
in  the  usual  way  by  induction  shocks,  and  he  takes  their  elec- 
trical response  as  his  guide  to  their  activity.  He  has  in  this 
way  studied  the  influence  of  numerous  reagents  and  drugs  upon 
nerve,  and  the  presence  and  extent,  or  the  absence  of  the 
galvanometric  answer  show  whether  any  particular  reagent 
increases,  diminishes,  or  annuls  nervous  action.  Among  the 
reagents  which  he  thus  investigated,  carbonic  acid  is  one,  and 
his  results  with  this  gas  are  most  instructive.  Large  doses  of 
carbonic  acid  act  like  an  anaesthetic,  and  completely  abolish 
the  electrical  response,  but  the  nerve  soon  recovers  when  the 
poisonous  gas  is  replaced  by  air.  Very  small  doses  of  carbonic 
acid  increase  its  activity,  and  the  swing  of  the  galvanometer 
needle  is  increased  when  the  nerve  is  thrown  into  action.  A 
nerve  thus  forms  a  very  delicate  test  object  for.  this  gas  ;  fa.r 


vii. J  KATABOLISM  OF  LECITHIN  83 

more  delicate,  in  fact,  than  most  chemical  reactions  are. 
When  a  nerve  is  excited  to  activity,  the  electrical  responses 
improve  ;  just  as  when  a  muscle  is  made  to  undergo  a  succes- 
sion of  contractions,  the  beneficial  effect  of  contraction 
manifests  itself  by  what  is  technically  called  the  "staircase 
phenomenon."  This  beneficial  effect  of  previous  action  is 
exactly  similar  to  what  is  produced  by  minute  doses  of 
carbonic  acid  gas,  and  Dr  Waller  argues  from  his  experiments 
that  they  prove  what  cannot  be  directly  tested  by  the  rougher 
methods  of  chemical  analysis,  namely,  that  activity  is  associated 
with  the  discharge  of  carbon  dioxide.  I  shall  have  to  return  to 
this  question  in  our  subsequent  discussion  of  fatigue. 

Evidence  of  Metabolic  Activity  in  Nervous  Structures  derived 
from  the  Examination  of  Cerebro- Spinal  Fluid,  and  of  Saline 
Extracts  of  Nervous  Tissues 

We  are  so  accustomed  to  associate  the  word  metabolism  with 
the  activity  of  the  proteid  constituents  of  protoplasm,  that  we 
are  sometimes  apt  to  forget  that  other  materials  frequently 
exhibit  a  similar  alternate  or  simultaneous  series  of  anabolic 
and  katabolic  phases.  In  nervous  structures  this  is  particularly 
true  for  their  complex  phosphorised  molecules.  Even  those 
who  like  Thudichum  have  approached  the  question  from  the 
purely  chemical  standpoint,  have  drawn  attention  to  the  lability 
of  lecithin.  Gumprecht  has  shown  that  perfectly  normal 
cerebro-spinal  fluid  contains  minimal  traces  of  choline,  a  sub- 
stance derived  from  the  decomposition  of  lecithin,  and  other 
phosphorised  fats.  This  trace  of  choline  represents  the  small 
balance  on  the  wrong  side  of  the  account.  This  difference 
becomes  much  more  pronounced  in  diseased  conditions.  This 
point  I  am  reserving  for  fuller  study  in  a  future  lecture. 

Exactly  similar  evidence  is  obtained  by  making  saline  ex- 
tracts of  perfectly  fresh  nervous  tissues.  Violent  reagents  which 
break  up  the  nervous  tissues  will  naturally  lead  to  the  appearance 
of  large  quantities  of  choline  in  the  extract  mixed  with  numerous 
other  substances.  But  physiological  saline  solution,  the  most 
harmless  of  all  reagents,  will  even  at  room  temperature  extract 


84 


METABOLISM  IN  NERVOUS  TISSUES 


[LECT. 


choline  from  perfectly  fresh  tissues  in  sufficient  quantities  to 
render  its  detection  by  both  chemical  and  physiological  tests  a 
comparatively  easy  task.  I  do  not  wish  to  dwell  at  this  point 
on  the  methods  adopted  for  the  detection  of  choline.  That  we 
shall  go  into  quite  fully  later.  I  will  only  say  that  its  most 
characteristic  physiological  action  when  injected  into  the  blood 
stream  of  an  anaesthetised  animal,  is  a  fall  of  blood  pressure  ; 
when,  however,  the  animal  has  received  a  previous  dose  of 
atropine,  the  fall  is  absent,  or  may  be  replaced  by  a  rise  of 
pressure  when  choline  is  injected.  This  is  illustrated  by  the 
next  two  figures  (Figs.  4  and  5). 


FIG.  4. 


FIG  5. 


Fig.  4. — Effect  on  injecting  extract  of  cat's  brain  before  atropine. 

Fig.  5. — Effect  after  atropine  of  the  same  extract  in  the  same  animal  (cat)  ;  the  fall 

of  blood  pressure  seen  in  Fig.  4  is  here  replaced  by  a  slight  rise.     With  pure 

solutions  of  choline  the  rise  is  generally  more  marked. 
Both  these  figures  are  considerably  reduced  in  size  ;  the  amount  of  reduction  can  be 

judged  from  the  measure  of  the  height  of  blood  pressure  indicated  in  millimetres 

on  the  side  of  Fig.  4. 

This  and  all  subsequent  tracings  are  to  be  read  from  left  to  right. 
The  time  tracing  marks  seconds. 
The  rising  of  the  signal  (lowest)  line  indicates  the  period  during  which  the  injection 

was  being  made  into  the  external  jugular  vein. 

The  presence  of  choline  in  these  extracts  is  a  very  positive 
sign  of  chemical  activity  in  the  living  nerve  structures  ;  some  of 
the  phosphorised  fat  has  undergone  katabolic  changes.  In 
addition  to  this  fact  there  is  a  further  one,  namely,  that  the  most 


vii j  FATIGUE  CHANGES  IN  NERVE-C&LLS  8$ 

active  part  of  the  nervous  sytem,  the  grey  matter,  yields  most 
choline  to  solvents. 

I  originally  took  up  the  subject  of  the  physiological  effects  of  injecting 
extracts  of  brain  and  other  nervous  tissues  *  in  consequence  of  a  paper  by 
Cleghorn.t  He  found  that  extracts  of  sympathetic  ganglia  produced  a  fall 
of  blood  pressure,  and  stated  that  extracts  of  other  nervous  tissues  did  not 
behave  in  this  way.  As  this  seemed  to  me  very  remarkable,  I  repeated  his 
experiments  ;  Osborne  and  Vincent  {  did  so  also,  and  we  both  found  that 
all  nervous  tissues  behave  similarly.  Substances  that  produce  a  fall  of 
blood  pressure  are  obtained  in  extracts  of  many  tissues,§  but  in  the  majority 
of  cases,  these  "  depressor  "  substances  have  not  been  chemically  identified. 
It  is,  however,  probable  that  in  some  instances,  the  depressor  substance  is 
inorganic  ;  potassium  and  ammonium  chloride  both  produce  a  fall  of  blood 
pressure,  which  is  unaffected  by  atropine.  The  question  is,  Are  there  any 
other  depressor  substances  besides  choline  in  extracts  of  nervous  tissues  ? 
Vincent  and  Cramer  ||  have  shown  that  there  are  ;  the  choline  exists  according 
to  them  in  the  condition  of  di-choline  anhydride  (which  is  a  small  refine- 
ment of  no  importance),  and  being  soluble  in  absolute  alcohol  can  be  sepa- 
rated almost  completely  from  the  inorganic  depressor  materials  (ammonium 
and  potassium  chlorides).1F 

Evidence  of  Metabolic  Activity  of  Nervous  Centres  derived  from 
Histological  Examination  of  Nerve-Cells 

We  know  that  in  a  muscle-nerve  preparation,  fatigue  is  due 
to  the  accumulation  of  the  products  of  muscular  activity,  and 
that  it  may  be  artificially  induced  by  irrigating  such  a  prepara- 
tion with  a  dilute  solution  of  sarcolactic  acid,  and  removed  by 
neutralising  this  with  salt  solution  containing  a  little  alkali.  It 
has  been  further  shown  that  the  muscular  fibres  are  not  to  any 

*  Jour,  of  Phys.,  vol.  xxvi.,  p.  229. 

t  American  Jour,  of  Phys.,  vol.  ii.,  p.  471.  Jour,  of  the  Boston  Soc.  of 
Med.  Sciences,  vol.  iv.,  p.  289. 

\  Jour,  of  Phys.,  vol  xxv.,  p.  283. 

§  Vincent  and  Sheen,  ibid.,  vol.  xxix.,  p.  242. 

||   Ibid.,  vol.  xxx.,  p.  143. 

IT  Read  also  in  this  connection  Gulewitsch,  Zeit.  f.  physiol.  Chem.,  vol. 
xxvii.,  p.  50.  Gumprecht,  loc.  cit.  Hunt,  Proc.  Amer.  Phys.  Soc.,  1899. 
The  veratrine-like  action  on  voluntary  muscles  described  by  Cleghorn  in  his 
extracts  of  sympathetic  ganglia  is  due  not  to  choline,  but  to  the  glycerin  he 
used  as  the  extracting  agent  (Lyle,  Proc.  Phys.  Soc.,  1901,  p.  xxvi.  ;  Jour,  of 
Phys.,  vol.  xxvi.). 


86  METABOLISM  IN  NERVOUS  TISSUES  [LECT. 

great  extent  the  seat  of  fatigue,  and  that  nerve-fibres  are 
inexhaustible.  By  a  process  of  exclusion,  the  seat  of  exhaustion 
has  thus  been  localised  in  the  intra-muscular  nerve-endings. 
But  when  a  muscle  is  fatigued  in  the  intact  body,  there  is 
another  factor  to  be  considered  beyond  the  mere  local  poisoning 
of  the  end-plates.  This  is  the  effect  of  the  products  of  muscular 
katabolism  passing  into  the  circulation  and  poisoning  the  central 
nervous  system.  It  is  stated  that  the  introduction  of  the  blood 
from  a  fatigued  animal  into  the  cerebral  circulation  of  a  second 
animal  will  produce  in  the  latter  all  the  signs  of  fatigue.  The 
blood  still  remains  alkaline  ;  the  toxic  material  cannot  therefore 
be  free  lactic  acid,  and  lactates  do  not  produce  the  effect. 
Mosso  has  advanced  the  theory  that  the  poisonous  substance  or 
substances  are  basic,  but  we  have  really  no  accurate  knowledge 
of  their  nature. 

There  is  considerable  discussion  just  now  in  the  scientific 
world  on  the  relative  importance  of  central  and  peripheral 
fatigue.  The  workers  at  the  Brussels  school  maintain  that  the 
peripheral  factor  is  the  more  important.  Those  at  Turin  under 
Mosso  are  prominent  adherents  of  the  doctrine  of  central 
fatigue. 

Many  years  ago,  Waller*  invented  an  instrument  which  he 
called  the  dynamograph.  It  consists  of  a  handle,  which  is  pulled 
up  by  the  hand  at  regular  intervals  against  a  strong  spring ; 
the  amount  of  movement  is  recorded  by  a  writing  lever,  upon  a 
slowly  revolving  drum.  Mosso  some  years  later  invented  a 
modification  of  this  instrument,  in  which  the  movements  of  a 
finger  in  raising  a  weight  are  similarly  recorded.  Since  then 
several  modifications  of  the  ergograph  (to  adopt  Mosso's  term) 
have  appeared,  f  Those  who  have  worked  with  such  instru- 
ments conclude  that  diminished  voluntary  power  occurs  at  a 
time  when  the  excitation  of  nerve  or  muscle  gives  no  sign  of 
ordinary  fatigue  at  the  periphery. 

*  Brit.  Med.Jour.,  25th  July  1885. 

t  At  the  actual  lecture  at  the  University  of  London,  some  of  these  instru- 
ments (Waller's,  Mosso's,  Cattell's,  and  Porter's)  were  exhibited.  Dr  Waller 
was  kind  enough  to  submit  himself  to  an  actual  experiment  carried  out  with 
his  own  instrument. 


vii.]  CHROMA  TOL  YSIS  87 

Among  the  recently  introduced  methods  of  examining  nerve- 
cells,  that  of  Golgi  (the  silver-chrome  process),  and  of  Nissl  (the 
methylene-blue  process)  stand  out  in  special  prominence.  The 
question  we  have  now  to  ask  is  whether  these  micro-chemical 
methods  show  any  changes  in  nerve-cells  as  a  result  of  activity. 
If  they  do,  we  have  a  most  valuable  piece  of  evidence  in  favour 
of  the  view  that  fatigue  in  the  central  nervous  system  is  an 
important  factor  in  the  causation  of  what  after  all  is  a  complex 
phenomenon,  which  is  doubtless  produced  in  several  ways.  Of 
the  two  methods  mentioned,  Golgi's  is  useless  for  the  purpose  ; 
the  methylene-blue  reaction  is  far  more  delicate,  and  is  the  only- 
one  which  is  really  helpful  from  this  point  of  view. 

We  need  hardly  discuss  the  question  whether  the  granules 
called  after  Nissl  are  present  as  such  in  healthy  nerve-cells,  or 
are  produced  by  the  alcohol  used  in  the  preliminary  hardening. 
Healthy  nerve-cells,  fixed  and  stained  in  a  constant  manner,  form 
the  equivalent  of  such  cells  during  life.  It  follows  that  if  cells 
prepared  by  the  same  method  show  a  difference  from  the 
equivalent  or  symbol  of  healthy  cells,  the  difference  is  a  measure 
of  some  change  that  has  occurred  during  life. 

Chromatolysis  is  the  term  applied  to  designate  the  disappear- 
ance or  disintegration  into  fine  dust-like  particles  of  these 
granules.  Micro-chemical  methods  have  shown  that  they  con- 
sist of  nucleo-proteid.  The  name  chromoplasm  is  given  to  this  ^ 
material  on  account  of  its  affinity  for  basic  dyes,  like  methylene 
blue.  The  name  kinetoplasm  was  given  to  it  by  Marinesco  in 
order  to  express  the  idea  that  it  forms  a  source  of  energy  to  the 
cell.  It  can  hardly  be  denied  that  the  substance  of  which  the 
granules  are  composed,  forming  as  it  does  so  large  a  proportion 
of  the  cell  contents,  and  made  of  a  material  in  which  nuclein 
is  an  important  constituent,  is  intimately  related  to  the  nutri- 
tional condition  of  the  neuron. 

Chromatolysis  generally  begins  at  the  periphery  of  the  cell  x, 
and  in  the  dendrons,  but  in  advanced  cases  the  whole  cell  may 
be  affected.  It  occurs  in  various  abnormal  states,  and  under 
the  influence  of  certain  poisons,  and  its  occurrence  indicates  a 
diminution  of  the  vital  interaction  between  the  highly  phosphor- 
ised  nucleus  and  the  surrounding  protoplasm.  Chromatolysis 


88  METABOLISM  IN  NER  VO  US  T1SS  UES  [LECT. 

alone,  however,  is  not  indicative  of  cell  destruction,  and  the  cell 
may  recover  its  functions  later  when  the  abnormal  condition 
passes  off.  The  integrity  of  the  nucleus  and  of  the  fibrils  is 
much  more  important  to  the  actual  vitality  of  the  cell. 

When  a  nerve-fibre  is  cut  across,  the  distal  segment  under- 
goes the  acute  change  known  as  Wallerian  degeneration.  But 
the  body  of  the  nerve-cell  and  the  piece  of  the  nerve-fibre  still 
attached  to  it  do  not  remain  unaffected ;  they  undergo  a  slow 
chronic  wasting,  and  one  of  the  earliest  signs  of  this  disuse- 
_  atrophy  is  chromatolysis. 

Chromatolysis,  therefore,  is  a  sign  of  inactivity ;  is  it  also  a 
sign  of  excessive  activity  ?  The  answer  to  this  question  is  an 
affirmative  one  according  to  most  observers. 

The  chromatoplasm  has  been  compared  to  the  granular 
material  present  in  secreting  cells  ;  in  such  cells,  before  secretion 
occurs,  the  granules  accumulate,  and  during  the  act  of  secretion 
they  are  discharged  and  converted  into  certain  constituents  of 
the  secretion.  In  a  somewhat  similar  way,  the  Nissl  granules 
are  used  up  during  the  discharge  of  energy  from  the  nerve-cells, 
and  this  may  be  regarded  as  a  visible  sign  of  fatigue.  The 
following  are  some  of  the  principal  observations  which  bear  out 
this  statement. 

Eve*  excited  the  cervical  sympathetic  nerve  of  the  rabbit  for 
twelve  hours,  and  he  found  in  the  upper  cervical  ganglion  that 
the  cells  presented  a  diffuse  staining  with  methylene  blue,  which 
he  attributes  to  the  formation  of  acid  substances. 

A  blue  stain  of  similar  appearance  may  be  induced  in  the 
motor  cells  of  the  spinal  cord  after  exhaustion  is  produced  in 
-^them  by  giving  strychnine.  Max  Verwornf  places  carbon 
dioxide  in  the  first  rank  of  the  fatigue  products,  the  accumulation 
of  which  leads  to  this  result.  It  is  probable  that  there  are  other 
fatigue  products  also,  for  after  strychnine  the  grey  matter  of  the 
cord  is  as  a  rule  acid  to  litmus  paper. 

If  the  nerve-cells  are  examined  after  a  prolonged  epileptic 
fit  in  which  there  has  been  a  very  massive  discharge  of  impulses, 
again  chromatolysis  is  found.  Some  neurologists  doubt  whether 

*  Jour,  of  Phys.)  vol.  xx.,  p.  334. 

t  Arch.fiir(Anat.  «.)  Phys.,  1900,  p.  132. 


M 


FIG.  6. — NISSL'S  GRANULES. 

A.  Normal  pyramidal  cell  of  human  cerebral  cortex. 

B.  Swollen  oedematous  pyramidal  cell  from  a  case  of  status  epilepticus.     Notice  diffuse  stain- 

ing and  breaking-up  of  Nissl's  granules  into  fine  particles  ;  the  nucleus  is  enlarged  and 
eccentric.  The  lymph  space  around  the  cell  is  enlarged. 

C.  Pyramidal  cell  of  dog  after  ligature  of  vessels  going  to  the  brain,  and  consequent  anaemia. 

Notice  the  great  swelling  of  the  nucleus  and  advanced  chromatolysis,  most  marked  at 
the  periphery  of  the  cell.  Figs.  A,  B,  and  C  are  after  Mott ;  magnification  in  each  case, 
700  diameters. 

[).  Skeleton  condition  of  cerebral  cell  in  a  rabbit,  produced  by  six  hours'  anaesthesia  with  ether. 
After  HAMILTON  WRIGHT. 

[To  face  page  §9. 


VIL]  ABSENCE  OF  FATIGUE  IN  NERVES  89 

this  is  associated  with  intense  activity,  or  whether  it  is  not 
wholly  or  in  part  caused  by  venosity  of  the  blood.  The  cells  are 
very  sensitive  to  altered  vascular  conditions ;  anaemia,  for  < 
instance,  produces  a  similar  change,  accompanied  by  swelling  of 
the  cell,  and  swelling,  and  in  extreme  cases  extrusion,  of  the 
nucleus. 

Some  very  striking  observations  have  been  made  on  bees  ; 
the  nerve-cells  of  animals  early  in  the  morning  have  been  com- 
pared with  those  of  others  in  the  evening  after  a  hard  day's 
work ;  the  very  extensive  chromatolysis  which  is  noticeable  in  - 
the  evening  animals  is  a  very  conclusive  piece  of  evidence  in 
favour  of  the  view  that  the  nerve-cells  afford  visible  evidence  of 
fatigue  changes. 

The  drawings  in  the  accompanying  plate  illustrate  some  of 
these  appearances  (Fig.  6).  A  is  a  normal  cell  showing  the 
Nissl  granules  intact.  B  is  a  cell  which  shows  chromatolysis 
as  the  result  of  the  epileptic  state  ;  C  shows  much  the  same  con- 
dition as  a  result  of  cerebral  anaemia.  D  illustrates  the  effect  of 
prolonged  narcosis,  which  we  shall  refer  to  later  in  the  course  of 
the  present  lecture. 

Fatigue  is,  therefore,  demonstrable  in  the  nerve-centres,  and 
in  the  peripheral  endings  of  nerve-fibres.  Which  is  the  more 
important  of  the  two,  I  do  not  pretend  to  be  able  to  decide 
absolutely  on  the  evidence  at  present  available ;  but  having 
examined  the  evidence,  I  am  inclined  to  take  the  view  that 
central  fatigue  is  the  more  important,  and  is  more  readily  - 
produced. 

The  Absence  of  Fatigue  Changes  in  Nerve-Fibres 

Not  the  least  interesting  of  the  facts  we  have  mentioned  is 
the  non-fatigability  of  nerve-fibres.  The  experiments  on  which 
the  assertion  rests  have  nearly  all  been  performed  with  medul- 
lated  motor  fibres.  The  method  adopted  in  such  experiments 
has  been  to  excite  the  nerves  for  a  number  of  hours,  and  to 
exclude  fatigue  in  the  terminal  structures  by  preventing  the 
impulses  reaching  the  peripheral  organ.  On  removing  the 
block  by  means  of  which  this  is  accomplished,  the  activity  of 


or  THI 
UNIVERSITY 


90  METABOLISM  IN  NERVOUS  TISSUES  [LECT. 

the  peripheral  organ  is  on  stimulation  of  its  nerve  still  mani- 
fested with  undiminished  force.  The  blocks  employed  have  been 
curare,*  a  galvanic  current/]-  the  application  of  ether,}  and  in  the 
case  of  secretory  fibres,  atropine.§ 

A  few  investigators  have  employed  non-medullated  fibres  in 
their  experiments.  In  his  experiments  on  the  cervical  sympa- 
thetic, Eve  1 1  found  that  the  vaso-constrictor  apparatus  in  the  ear 
vessels  was  still  in  action  at  the  end  of  twelve  hours*  excitation. 
Here  no  block  was  used,  for  in  the  vaso-motor  nerves  fatigue  of 
even  the  peripheral  endings  does  not  occur  in  demonstrable 
amount. 

Howell,  Budgett,  and  Leonard  11  state  that  vaso-constrictor 
and  cardio-inhibitory  fibres  show  no  functional  fatigue ;  but  as 
the  longest  time  during  which  they  applied  continuous  excitation 
was  only  one  hour,  this  contention  can  hardly  be  considered  to 
be  satisfactorily  proved. 

What  does  this  mean  ?  I  take  it  that  it  does  not  mean  that 
the  nerve-fibres  undergo  absolutely  no  metabolic  changes  when 
transmitting  a  nerve  impulse.  It  means  that  the  change  is  so 
slight,  and  the  possibilities  of  repair  so  great,  that  fatigue  in  the 
usual  acceptation  of  the  term  cannot  be  demonstrated.  This  is 
an  illustration  of  the  wonderfully  economic  way  in  which  Nature 
often  works. 

That  a  change  does  occur  in  a  nerve-fibre  is  evidenced  by  the 
electrical  variation  it  undergoes  and  which  can  be  detected  by 
the  galvanometer  or  the  electrometer.  Further,  we  have  already 
seen,  Waller  believes  he  has  shown  that  carbonic  acid  is  evolved 
by  the  axis-cylinder.  How,  then,  can  we  account  for  the  fact 
that  fatigue  cannot  be  shown  to  occur  ?  To  meet  this  difficulty 
Waller  tentatively  suggested  a  most  ingenious  explanation, 
which  it  will  be  well  to  give  in  his  own  words.**  He  says  :  "  I 

*  Bowditch,  Jour,  of  Phys.,  1885,  vol.  vi.,  p.  133. 

t  Berustein,  Pfliiger's  Arthtvt  1877,  p.  289;  Wedenski,  Centralbl.  f.  d. 
med.  Wissensch.,  1884,  vol.  xxii.,  p.  65. 

t  Maschek,  Sitzungsb.  d.  k.  Akad.  d.  Wissensch.,  Wien,  1887,  vol.  xcv., 
Abth.  3,  p.  109. 

§  Lambert,  Compt.  rend.  Soc.  Biol.,  tenth  series,  1894,  vol.  i.,  p.  511. 

||  Loc.  cit.  11"  Jour,  of  Phys.,  1894,  vol.  xvi.,  p.  298. 

**  Lectures  on  Physiology,  first  series,  Animal  Electricity,  1897,  p.  70. 


vn.]     EXPERIMENTS  ON  NON- MED  ULLA  TED  NERVES        91 

wonder  does  this  carbonic  acid  become  altogether  dissipated ; 
may  it  not  perhaps  be  reinvolved  in  some  storage  combination, 
as  the  nerve-fat,  perhaps,  that  is  so  prominent  a  constituent  of 
fully  evolved  nerve.  Such  nerve  consists  of  proteid  axis  and 
fatty  sheath ;  the  axis — which  is  the  offshoot  of  a  nerve-cell — 
is  the  specially  conductile  part,  the  sheath  is  a  developmental 
appendix,  not  directly  connected  with  any  nerve-cell.  Yet,  cut 
the  nerve,  and  sheath  as  well  as  axis  undergo  Wallerian 
degeneration,  which  is  evident  proof  of  a  functional  commerce 
between  sheath  and  axis.  All  these  things  to  my  mind  reconcile 
themselves  with  the  notion  that  the  active  grey  axis  both  lays 
down  and  uses  up  its  own  fatty  sheath,  and  that  it  is  inex- 
haustible not  because  there  is  little  or  no  expenditure,  but 
because  there  is  an  ample  re-supply." 

A  year  or  two  after  these  words  were  written,  Miss  Sowton,* 
at  Dr  Waller's  suggestion,  undertook  a  piece  of  work  in  order  to 
test  the  truth  of  this  hypothesis.  If  the  absence  of  fatigue  is 
due  to  the  presence  of  the  fatty  sheath,  fatigue  ought  to  be 
demonstrable  in  nerve-fibres  in  which  the  fatty  sheath  is  absent 
She  selected  the  olfactory  nerve  of  the  pike  as  the  non- 
medullated  nerve  with  which  to  try  the  experiment,  and  her 
results  confirmed  Waller's  expectation ;  the  galvanometric 
replies  of  the  nerve  become  somewhat  feebler  after  repeated 
stimulation. 

It  appeared  to  me  advisable  to  test  the  question  in  another 
way.  Some  doubt  has  recently  been  cast  on  the  trustworthiness 
of  the  electrical  response  as  a  sign  of  nervous  activity.^  As 
the  doubt  has  arisen,  the  greater  becomes  the  necessity  for  a 
fresh  method  of  attacking  the  problem.  The  splenic  nerves 
appeared  to  be  the  most  convenient  large  bundles  of  non- 
medullated  fibres  for  the  purpose.  Dr  T.  G.  Brodie  was  associ- 
ated with  me  in  carrying  out  the  investigation.  A  dog  is 
anaesthetised  with  morphine  and  A.C.E.  mixture,  the  abdomen 
opened,  the  spleen  exposed,  and  the  splenic  nerves  which  lie  by 
the  side  of  the  main  splenic  artery  are  laid  bare.  It  is  quite 

*  Proc.  Roy.  Soc.,  1900,  vol.  Ixvi.,  p.  379. 

t  See  Professor  Gotch's  article  "Nerve"  in  Schafer's  Text-book  of 
Physiology. 


METABOLISM  IN  NERVOUS  TISSUES 


[LECT. 


easy  to  dissect  out  a  length  of  nerve  sufficient  for  the  experi- 
ment (\\  to  2  inches).  The  nerve  is  then  cut  as  far  from  the 
spleen  as  possible,  and  the  spleen  is  enclosed  in  an  oncometer, 
similar  to  that  employed  by  Schafer  in  his  work  on  the  spleen.* 
On  stimulating  the  nerve  with  a  weak  faradic  current  the  organ 
contracts,  and  the  recording  lever  falls.  The  diminution  of  the 
size  of  the  spleen  is  quite  visible  to  the  naked  eye,  however, 
without  the  use  of  any  apparatus.  The  next  thing  to  do  is  to 
put  a  block  on  the  course  of  the  nerve,  which  will  prevent  the 
nerve  impulses  from  reaching  the  spleen.  Here  we  met  with 
some  difficulty.  Curare  and  atropine  are  both  ineffective ;  the 
constant  current  has  a  great  disadvantage ;  non-medullated 


G.P. 


FlG.  7. — Apparatus  for  obtaining  splenic  curves.  S,  spleen  in  oncometer  O,  which  is 
made  of  guttapercha,  and  covered  with  a  glass  plate  (G.P.)  luted  on  with 
vaseline.  M  is  the  splenic  mesentery,  containing  vessels  and  nerves  ;  this  passes 
through  a  slit  in  the  base  of  the  oncometer,  and  is  made  air-tight  with  vaseline. 
The  oncometer  is  connected  to  one  of  Brodie's  bellows  recorders  (B)  by  the  india- 
rubber  tube  (R),  the  side  tube  (T)  being  closed  during  an  experiment  by  a  piece 
of  glass  rod.  The  recording  lever  (L)  writes  on  a  revolving  drum. 

nerves  are  so  much  affected  that  very  feeble  constant  currents 
(one-third  of  a  Daniell  cell)  will  completely  block  the  transmis- 
sion of  impulses,  and  not  only  that,  but  the  nerve  remains 
blocked  after  the  current  is  removed.  After  the  current  has 
been  allowed  to  flow  for  two  minutes  the  nerve  remains  impass- 
able to  nerve  impulses  for  an  hour  or  more,  and  then  slowly 

*  Schafer  and  Moore,  Jour,  of  Phys.^  vol.  xx.,  p.  I. 


vii.]     EXPERIMENTS  ON  NON-MEDULLATED  NERVES        93 


DT 


recovers.  If,  therefore,  faradic  excitation  of  the  nerve  is  kept 
up  all  this  time  and  fails  to  excite  the  contraction  of  the  spleen 
after  the  removal  of  the  constant  current,  it  is  impossible 
to  say  whether  this  is  due  to  fatigue  of  the  nerve-fibres  on 
the -proximal  side  of  the  block,  or  whether  it  may  not  be  due 
to  the  fact  that  the  block  created  by  the  constant  current  is 
still  effective. 

Our  best  results  were  obtained  by  using  cold  instead  of  a 
constant  current  as  our  blocking  agent. 

Fig.  7  is  an  outline  drawing  of  the  apparatus  used. 

Fig.  8  shows  the  arrangement  adopted  in  connection  with 
the  nerve.  The  nerve  (N)  rests  on  a  metal  tube  (T)  through 
which  fluid  can  be  kept  flowing.  E  is 

the  situation  of  the  electrodes.     If  the       ^ -g ^ 

nerve  is  excited,  the  spleen  contracts 
and  the  recording  lever  (in  Fig.  7)  falls. 
If  now  brine  at  o°  to  2°  C.  is  kept  flow- 
ing through  T,  the  nerve  impulses  are 
blocked  by  the  cold,  and  cannot  reach 
the  spleen.  Immediately  the  cold  brine 
is  replaced  by  warm  water  at  30°  C.,  the 
nerve  again  becomes  passable  by  nerve 
impulses,  and  the  spleen  contracts  once 
more. 

If  now  the  water  in  T  is  kept  at  the 
low  temperature  mentioned,  and  the 
nerve  is  being  excited  with  strong  in- 
duction shocks  all  the  time,  the  spleen 
remains  irresponsive ;  the  nerve-im- 
pulses are  able  to  reach  T  but  not  to 
pass  it.  If  then  warm  water  is  passed 
through  T,  and  the  block  produced  by 
the  cold  is  thus  removed,  and  the  spleen  continues  to  be  irre- 
sponsive, we  have  a  proof  that  the  piece  of  nerve  between  E 
and  T  has  been  fatigued.  But  our  experiments  have  shown 
us  that  non-medullated  nerve  is  just  as  difficult  to  fatigue 
as  medullated  nerve.  Even  after  six  hours'  continuous  excita- 
tion the  nerve  is  just  as  excitable  as  it  was  at  the  start,  and 


FlG.  8. — Arrangement  of  ap- 
paratus in  connection  with 
the  splenic  nerve.  S  is  the 
spleen,  and  N  the  main 
bundle  of  nerves.  The 
nerve  rests  on  the  metal 
tube  (T),  through  which 
water  at  the  required  tem- 
perature is  kept  flowing, 
and  on  the  electrodes  (E), 
which  come  from  the 
secondary  coil  of  an  in- 
ductorium. 


94  METABOLISM  IN  NERVOUS  TISSUES  [LECT. 

a  full  splenic  contraction  is  obtained  when  the  cold  block  is 
removed. 

At  the  lecture  an  experiment  of  this  kind  was  shown  to  those  present ; 
and  it  may  not  be  uninteresting  to  reproduce  the  tracings  which  were 
actually  taken  (at  the  University  of  London).  At  the  commencement  of 
the  lecture,  tracing  B  (Fig.  9),  was  obtained  by  stimulating  one  of  the  large 
splenic  nerves.  The  downward  movement  of  the  lever  began  very  soon 
after  the  faradic  excitation  started  (the  duration  of  which  is  shown  by  the 
raising  of  the  signal  line) ;  recovery  began  a  little  later,  and  finally  the 
splenic  lever  wrote  at  its  former  level.  The  cold  block  was  then  introduced, 
and  faradic  excitation  was  maintained  until  the  end  of  the  lecture,  the 
spleen  being  irresponsive  all  the  time.  At  the  end  of  the  lecture,  about  an 
hour  and  a  half  later,  the  cold  brine  was  replaced  by  warm  water ;  during 
this  manipulation  the  excitation  was  stopped  for  a  few  moments  ;  then 
excitation  produced  the  contraction  seen  in  tracing  C,  which  is  equal  in 
extent  to  that  in  B.  It  did  not  last  quite  so  long,  simply  because  the 
excitation  was  a  shorter  one  ;  the  excitation  was  of  the  same  strength 
(Daniell  cell  attached  to  primary  coil,  secondary  coil  of  the  du  Bois-Reymond 
inductorium  pushed  right  home)  in  both  cases.  The  bellows  recorder 
possesses  the  advantage  of  being  capable  of  calibration  ;  the  contraction  of 
the  spleen  in  this  case  squeezed  into  the  circulation  about  20  c.c.  of  blood  ; 
this  caused  a  slight  temporary  increase  of  arterial  pressure,  a  tracing  of 
which  was  being  simultaneously  recorded  by  a  mercurial  manometer  attached 
to  the  carotid  artery.  It  is  a  wise  precaution  to  take  such  a  tracing,  because 
in  prolonged  experiments  of  this  kind,  the  general  condition  of  the  animal 
can  be  gauged  by  the  height  of  arterial  pressure.  I  have  not,  however, 
thought  it  worth  while  to  reproduce  this  part  of  the  record. 

A  possible  objection  to  this  experiment  might  be  made  in  this  way.  It 
is  well  known  that  heart  muscle  obeys  the  rule  which  Waller  has  so  tersely 
termed  "all  or  nothing."  That  is,  a  stimulus  strong  enough  to  provoke  a 
contraction  of  cardiac  muscle  always  calls  forth  a  maximum  contraction.  If 
the  same  rule  applies  to  the  smooth  muscle  of  the  spleen,  the  excitation 
shown  in  tracing  C  might  really  be  quite  a  weak  excitation  owing  to  nerve 
fatigue,  but  still  sufficient  to  evoke  a  full  splenic  reply.  Happily,  this 
objection  does  not  hold  ;  plain  muscle  does  not  obey  the  "all  or  nothing" 
rule  ;  within  fairly  wide  limits  the  amount  of  contraction  is  roughly 
proportional  to  the  strength  of  the  stimulus.  We  have  proved  this  over  and 
over  again  with  the  spleen.  During  the  rehearsal  of  this  very  experiment,  Dr 
Brodie  and  I  tried  various  positions  of  the  secondary  coil,  and  in  tracing  A 
is  shown  one  of  the  responses  to  a  weak  excitation.  We  finally  decided  to 
push  the  secondary  coil  right  home  for  the  lecture  experiment,  in  order  to 
make  the  result  as  striking  as  possible. 

In  addition    to   our  work    on   the  spleen,  we  made  corre- 
sponding observations  on  the  vaso-motor  nerves  contained  in  the 


VIL]  STIMULATION  FATIGUE  95 

sciatic  nerve  of  the  dog,  the  volume  of  the  leg  being  recorded 
with  a  plethysmograph,  and  we  also  repeated  Eve's  experiments 
on  the  cervical  sympathetic  running  to  the  ear  of  the  rabbit. 

In  no  case  were  we  able  to  demonstrate  any  functional 
fatigue.  But  we  did  notice,  especially  in  vaso-motor  nerves,  a 
phenomenon  which  Howell  terms  stimulation  fatigue  ;  this  means  ~ 
that  the  actual  spot  of  nerve  stimulated  becomes  after  a  time 
less  excitable,  and  finally,  inexcitable,  though  it  will  still 
transmit  impulses,  if  the  excitation  is  applied  above  the  spot 
originally  stimulated.  We  think  that  the  use  of  the  term 
"  fatigue "  in  this  connection  is  a  mistake ;  the  prolonged 
electrical  excitation  causes  injurious  polarisation  (due  to  electro- 
lytic changes)  of  the  nerve,  which  renders  it  less  excitable.  This 
view  has  been  confirmed  by  Professor  Gotch  by  means  of  experi- 
ments with  the  capillary  electrometer.  This  so-called  "  stimulation 
fatigue "  was  not  excluded  in  Miss  Sowton's  experiments,  and 
will  possibly  explain  her  results.  The  splenic  nerves,  curiously 
enough,  do  not  exhibit  this  phenomenon  in  any  marked  degree, 
and  so  were  peculiarly  well  adapted  to  test  the  question  of 
functional  fatigue.  On  a  priori  grounds  we  should  hardly 
expect  non-medullated  nerves  to  be  peculiarly  susceptible  of  real 
fatigue,  when  one  considers  how  many  of  them,  like  the  vaso- 
constrictors, are  in  constant  action  throughout  life. 

Our  final  conclusion  therefore  is,  that  although  fatigue  effects 
are  demonstrable  where  nerve-fibres  start  in  the  nerve-cells,  and 
also  where  they  terminate  in  peripheral  structures,  at  present 
fatigue  has  not  been  demonstrated  to  occur  in  the  course  of  the 
fibres  between  these  two  extremities.  Further,  that  although 
the  possibility  still  remains  that  this  is  to  be  explained  on 
Waller's  hypothesis  of  "functional  commerce"  between  the  axis- 
cylinder  and  its  sheath,  it  is  not  the  medullary  sheath  which  is 
essential,  for  fatigue  is  just  as  difficult  to  demonstrate  in  non- 
medullated  nerves  as  in  those  of  the  medullated  variety. 

The  experiment  demonstrated  at  the  University  of  London  led  Dr 
Waller  to  suggest  to  Dr  Alcock  that  he  should  investigate  the  behaviour  of 
the  splenic  nerve  towards  the  galvanometer,  and  he  has  since  then  pub- 
lished his  results  ;*  his  chief  conclusions  are  :— 

*  froc.  Roy.  See.,  1904,  vol.  Ixxiii.,  p.  1.66, 


96  METABOLISM  IN  NERVOUS  TISSUES  [LECT. 

1.  Non-medullated  nerves  exhibit  a  negative  variation  and  current   of 
injury  three  times  greater  in  magnitude  than  the  inedullated  nerves  of  the 
same  animal. 

2.  The  negative  variation  (or  current  of  action)  of  non-medullated  nerves 
undergoes  a  progressive  diminution  with  repeated  stimuli.     (Confirmatory 
of  Miss  Sowton.) 

3.  The  immediate  cause  of  this  diminution  is  a  localised  change  at  the 
point  of  excitation.     (Confirmatory  of  our  suggested  explanation  of  Miss 
Sowton's  results.) 

4.  The  electrotonic  currents  of  non-medullated  nerves  are  very  small, 
only  about  one-fortieth  of  those  in  medullated  nerves.     (This  explains  2  and 
3,  because  the  exciting  current  being  confined  to  the  place  of  application, 
has  a  greater  density,  and  therefore  a  greater  local  effect.) 

In  these  days  when  physiologists  are  so  prone  to  differences  of  opinion, 
it  is  satisfactory  that  two  such  diverse  methods  as  those  adopted  by  Alcock 
and  by  Brodie  and  myself,  have  led  to  the  same  conclusion  concerning  the 
absence  of  true  functional  fatigue  in  non-medullated  nerve-fibres. 

Sleep  and  Narcosis 

Having  now  studied  fatigue  in  many  of  its  aspects,  it  is 
appropriate  that  we  should  in  conclusion  turn  our  attention 
to  a  brief  consideration  of  sleep,  Nature's  great  restorer  for 
exhausted  "nerves." 

..  Theories  to  account  for  sleep  are  numerous,  and  none  are 
satisfactory.  Thus  by  some  it  has  been  attributed  to  changes 
in  the  blood-supply  of  the  brain,  and  ultimately  referred  to 
fatigue  of  the  vaso-motor  centres.  The  existence  of  an  effective 
vaso-motor  mechanism  in  the  cerebral  blood-vessels  themselves 
is  problematical ;  so  that  if  changes  occur  in  the  cerebral 
blood-pressure  or  rate  of  flow,  they  are  mainly  secondary  to 
those  which  are  produced  in  other  parts  of  the  body.  It  is, 
however,  quite  possible  that  the  vascular  condition  is  rather 
the  concomitant  or  consequence  of  sleep  than  its  cause. 

Some  of  the  theories  to  account  for  sleep  have  been  chemical. 
Thus  certain  observers  have  considered  that  sleep  is  the  result 
of  the  action  of  chemical  materials  produced  during  waking 
hours,  which  have  a  soporific  effect  on  the  brain  ;  according 
to  this  theory,  awakening  from  sleep  is  due  to  the  action  of 
certain  other  materials  produced  during  rest,  which  have  the 
opposite  effect.  Obersteiner  has  gone  so  far  as  to  consider 


VIL]  SLEEP  AND  NARCOSIS  97 

that  the  soporific  substances  are  reducing  in  nature,  and  others 
regard  them  as  alkaloidal.  These  theories  all  rest  upon  the 
slimsiest  foundations,  and  none  has  yet  been  found  to  stand 
experimental  tests. 

Then  there  are  what  we  may  term  histological  theories  of 
sleep,  and  these  are  equally  unsatisfactory.  The  introduction 
of  the  Golgi  method  opened  a  fresh  field  for  investigators, 
and  several  have  sought  to  find  by  this  method  a  condition 
of  the  neurons  produced  by  narcotics  like  opium  and  chloro- 
form, which  is  different  from  that  which  obtains  in  the  waking 
state. 

Demoor*  and  others  found  that  in  animals  in  which  deep 
anaesthesia  has  occurred,  that  the  dendrites  exhibit  moniliform 
swellings,  that  is,  a  series  of  minute  thickenings  or  varicosities. 
On  the  strength  of  this  observation,  he  has  formulated  what 
we  may  call  a  bio-physical  theory  of  sleep.  In  the  waking 
state,  the  neighbouring  nerve-units  are  in  contact  with  each 
other ;  transmission  of  nerve-impulses  from  neuron  to  neuron 
is  then  possible,  and  the  result  is  consciousness  ;  during  sleep 
the  dendrites  are  retracted  in  an  amoeboid  manner  ;  the  neurons 
are  therefore  separated,  and  the  result  is  unconsciousness. 

Lugaro,  on  the  other  hand,  takes  the  precisely  contrary 
view.  He  was  not  able  to  discover  monilifoVm  enlargements, 
and  his  bio-physical  hypothesis  is  that  the  interlacing  of  den- 
drites is  much  more  intimate  during  sleep  than  during  con- 
sciousness. He  therefore  explains  sleep  by  supposing  that  the 
definite  and  limited  relationships  between  neurons  no  longer 
exist,  but  are  lost  and  rendered  ineffective  by  the  universality 
of  the  connecting  paths.  It  is  not  very  difficult  to  explain 
such  divergence  of  views,  for  they  both  depend  mainly  on 
observations  made  by  a  single  method  ;  and  the  method  itself 
is  open  to  objection.  It  is  one  which  gives  even  in  the  same 
brain  most  inconstant  results,  and  is  not  calculated  to  show 
much  more  than  a  mere  outline  of  a  few  of  the  cells  and 
their  branches.  So  much  doubt  has  arisen  of  late  in  regard 
to  the  trustworthiness  of  the  method,  that  many  neurologists 
are  beginning  to  doubt  whether  the  neuron  theory  implying 
*  Arch,  de  Biol.,  1896,  vol.  xiv. 

G 


98 


METABOLISM  IN  NERVOUS  TISSUES 


[LECT. 


absolute  non-continuity  of  nerve-units  has  been  satisfactorily 
proved,  and  there  is  a  tendency  to  return  to  the  idea  of  a 
connecting  network  not  very  different  from  that  originally 
put  forward  by  Gerlach. 

A  more  satisfactory  investigation  of  the  effect  of  anaesthetics 
on  nerve-cells  has  been  carried  out  by  Dr  Hamilton  Wright,* 
who  performed  the  majority  of  his  experiments  in  my 
laboratory. 

He  used  rabbits  and  dogs,  and  subjected  them  to  ether 
and  chloroform  narcosis  for  periods  varying  from  half  an 


i- 


FlG.  10. — Moniliform  enlargements  on  dendrites  of  nerve-cells,  rendered  evident  by 
Cox's  method.  A  is  a  cortical  cell  of  a  rabbit ;  B  is  a  corresponding  cell  of 
a  dog's  brain,  after  six  hours'  anaesthetisation  with  ether  in  each  case. 
HAMILTON  WRIGHT. 

hour  to  nine  hours.  In  both  animals  he  found  that  the  nerve- 
cells  are  affected,  but  in  rabbits  much  more  readily.  This 
accords  quite  well  with  what  is  known  regarding  the  suscepti- 
bility of  rabbits  as  compared  to  dogs  towards  the  influence 
of  these  narcotising  agents.  In  a  rabbit  the  nerve-cells, 
especially  of  the  cerebrum,  show  changes  even  after  only 
half  an  hour's  anaesthesia,  but  in  dogs  at  least  four  hours' 
*  Jour,  of  Phys.,  1900,  vol.  xxvi.,  p.  30  ;  1901,  vol.  xxvi..  p.  362. 


VIL]  SLEEP  AND  NARCOSIS  99 

anaesthesia  must  be  employed.  By  the  Golgi  method  (Cox's 
modification)  the  moniliform  enlargements  can  be  seen.  These 
become  more  numerous,  larger,  and  encroach  more  and  more 
on  the  dendritic  stems,  the  longer  the  anaesthesia  is  kept 
up.  The  accompanying  illustrations  (Fig.  10)  show  the  appear- 
ances seen. 

Lugaro's  failure  to  find  these  appearances  is  doubtless  due 
to  his  not  having  maintained  the  anaesthesia  long  enough  in 
his  dogs. 

Wright  started  his  work  with  a  bias  in  favour  of  Demoor's 
bio-physical  theory,  but  he  soon  found  that  the  theory  was 
untenable ;  the  results  of  his  observations  have  shown  him 
that  the  action  of  anaesthetics  is  bio-chemical  rather  than  bio- 
physical, and  he  has  been  led  to  this  conclusion  by  the  em- 
ployment of  other  histological  methods,  particularly  the 
most  sensitive  one  we  possess,  namely,  the  methylene-blue 
reaction. 

Owing  to  the  chemical  action  of  the  anaesthetic  in  the  cells, 
the  Nissl  bodies  have  no  longer  an  affinity  for  methylene  blue, 
and  the  cells  consequently  present  what  Wright  calls  a  rarefied 
appearance ;  when  this  becomes  marked  the  cells  appear  like 
the  skeletons  of  healthy  cells  (see  Fig.  6,  D).  In  extreme  cases 
the  cells  look  as  though  they  had  undergone  a  degenerative 
change,  and  after  eight  or  nine  hours'  anaesthesia  in  dogs,  even 
the  nucleus  and  nucleolus  lose  their  affinity  for  basic  dyes.  The 
change,  however,  is  not  a  real  degeneration,  and  passes  ofT  when 
the  drug  disappears  from  the  circulation.  Even  after  nine 
hours'  anaesthesia  the  cells  return  rapidly  to  their  normal  con- 
dition, stain  normally,  moniliform  enlargements  have  dis- 
appeared, and  no  nerve-fibres  show  a  trace  of  Wallerian 
degeneration.  The  pseudo-degenerative  change  produced  by 
the  chemical  action  of  the  anaesthetic  no  doubt  interferes  with 
the  normal  metabolic  activity  of  the  cell-body,  and  this  produces 
effects  on  the  cell  branches.  In  the  early  stages  of  Wallerian 
degeneration,  the  branch  of  the  nerve-cell  which  we  call  the 
axis-cylinder  presents  swellings  or  varicosities,  produced  by 
hydration  or  some  similar  chemical  change.  The  moniliform 
enlargements  seen  during  the  temporary  pseudo-degenerative 


io6  METABOLISM  IN  NERVOUS  TISSUES  [LECT. 

effects  produced  by  anaesthetics  are  comparable  to  this.*  These 
enlargements  are  therefore  not  the  primary  cause  of  loss  of 
consciousness,  but  are  merely  secondary  results  of  changes  in 
the  cell-body.  When  a  tree  begins  to  wither,  the  earliest 
apparent  change  is  noticed  in  the  branches  most  remote  from 
the  centre  of  nutrition,  the  root ;  as  the  changes  in  the 
centre  of  nutrition  become  more  profound,  the  larger  branches 
become  implicated,  but  the  seat  of  the  mischief  is  not 
primarily  in  the  branches.  This  illustration  may  serve  to 
render  intelligible  what  is  found  in  nerve-cells  and  their 
branches. 

Whether  the  appearances  found  in  dogs  and  rabbits  are 
applicable  to  the  human  subject,  is  another  question.  I  am 
inclined  to  think  that  we  may  safely  regard  them  as  such  ; 
there  is  no  reason  why  an  anaesthetic  should  act  differently  in 
different  animals.  The  resistance  of  the  animal  is  a  variable 
factor,  and  this  causes  a  variation  in  degree  only ;  the  effect 
is  probably  the  same  in  kind  for  all  animals,  man  included. 

But  I  feel  that  we  should  be  very  chary  in  concluding  that 
the  artificial  sleep  of  a  deeply-narcotised  animal  is  any  criterion 
of  what  occurs  during  normal  sleep.  The  sleep  of  anaesthesia 
is  a  pathological  condition  due  to  the  action  of  a  poison.  The 
drug  reduces  the  chemico-vital  activities  of  the  cells,  and  is,  in  a 
sense,  dependent  on  an  increasing  condition  of  exhaustion, 
which  may  culminate  in  death.  Normal  sleep,  on  the  other 
hand,  is  not  produced  by  a  poison,  or  at  any  rate  we  have  no 
evidence  of  any  poison  ;  it  is  the  normal  manifestation  of  one 
•stage  in  the  rhythmical  activity  of  nerve-cells,  and  though  it 
may  be  preceded  by  fatigue  or  exhaustion,  it  is  accompanied 
by  repair,  the  constructive  side  of  metabolic  activity. 

Since  the  foregoing  lecture  was  delivered,  a  very  valuable  contribution  to 
the  already  extensive  literature  on  the  anaesthetic  question  has  been 
published  by  B.  Moore  and  H.  E.  Roaf.t  They  point  out  that  although 
anaesthetics  are  very  numerous,  there  is  probably  only  one  type  of  interaction 

*  Some  observers  look  upon  the  varicosities  as  artifacts.  If  they  are, 
they  ought  to  have  been  found  in  all  Wright's  specimens,  for  the  method  of 
preparation  was  the  same  throughout. 

t  Proc.  Roy.  Soc.,  1904,  vol.  Ixxiii.,  p.  382. 


VIL]  NARCOSIS  101 

between  the  anaesthetic  and  cell-protoplasm.  The  cells  usually  investigated, 
as  in  Hamilton  Wright's  work,  are  the  nerve-cells,  naturally  because  the 
state  of  quiescence  produced  in  those  cells  underlies  the  state  of  uncon- 
sciousness ;  but  all  other  cells  are  similarly  affected,  although  in  varying 
degree.  The  metabolic  processes  in  ciliated  epithelium,  amcebae,  bacteria, 
etc.,  are  stilled  as  effectually  as  those  in  nerve-cells  are.  Hence  the  action 
of  the  anaesthetic  must  be  due  to  a  change  in  some  substance  which  is 
uniformly  present  in  all  kinds  of  cell-protoplasm. 

From  this  wide  point  of  view,  anaemia  or  hyperaemia  of  the  brain 
described  by  different  observers  must  accordingly  be  set  down  as 
secondary  effects,  and  not  primary  causes,  of  anaesthesia.  Similarly, 
theories  based  on  the  high  content  in  lecithin,  cholesterin,  and  fatty  deriva- 
tives soluble  in  ether  or  chloroform,  of  the  nerve-cell,  cannot  furnish  any 
explanation  of  anaesthesia.  The  changes  noted  by  Wright  take  time  to 
develop,  and  form  a  signal  of  the  changes  produced  in  the  protoplasm  in 
a  marked  degree  by  the  prolonged  action  of  the  anaesthetic. 

The  most  constant  constituent  of  cell-protoplasm  is  proteid,  and  accord- 
ingly the  authors  turned  their  attention  in  the  experiments  they  have 
hitherto  recorded,  to  the  action  of  chloroform  on  proteid  material.  They 
find  that  unstable  compounds  of  proteid  and  chloroform  are  obtainable. 
In  this  way  chloroform  is  combined  with  the  blood-proteids,  and  accounts 
for  the  fact  that  chloroform  is  so  much  more  soluble  in  blood  than  in  water 
or  salt  solution.  The  chloroform-proteid  compound  is  compared  to  oxy- 
haemoglobin,  and  undergoes  dissociation  in  the  same  kind  of  way  ;  just  as 
oxyhaemoglobin  parts  with  its  oxygen  to  the  cells  of  the  tissues,  so  the 
chloroform  parts  company  from  the  blood-proteid,  and  enters  into  combina- 
tion with  the  proteid  matter  of  cell-protoplasm  ;  this  limits  the  activity  of 
the  cell-protoplasm,  and  produces  anaesthesia.  In  time,  when  the  adminis- 
tration of  the  anaesthetic  has  ceased,  and  the  chloroform  tension  in  the 
blood  is  no  longer  maintained,  the  combination  between  cell-proteid  and 
chloroform  dissociates,  and  anaesthesia  passes  off. 


LECTURE   VIII 

THE  COAGULATION  TEMPERATURE  OF  THE  NERVE- 
PROTEIDS,  AND  ITS  BEARING  ON  T.HE  QUESTIONS  OF: 
(l)  THE  GALVANOMETRIC  RESPONSE  OF  NERVE  UNDER 
VARYING  TEMPERATURES  ;  (2)  HEAT  CONTRACTION  IN 
NERVE  ;  AND  (3)  HYPERPYREXIA 

I  HAVE  already  told  you  something  about  the  proteids  of 
nervous  tissues  (pp.  61-63),  but  my  affection  for  questions  of 
proteid  chemistry  has  prompted  me  to  inflict  upon  you  a  whole 
lecture  to  be  devoted  to  certain  further  results  of  our  study  of 
these  substances.  These  come  under  three  heads  : — 

(1)  The   influence   of  temperature    on    the    galvanometric 
response  of  nerve  to  stimulation 

(2)  Heat  contraction  in  nerve. 

(3)  The   coagulation    temperature  of    cell-globulin    and    its 
bearing  on  hyperpyrexia. 

The  Influence  of  Temperature  on  the  Galvanometric  Response  of 
Nerve  to  Stimulation 

You  all  know  that  when  a  nerve  is  stimulated  to  activity,  it 
undergoes  a  change  of  electrical  potential,  which  can  be  detected 
with  the  galvanometer  or  electrometer.  This  we  still  call  by 
du  Bois-Reymond's  old  name,  the  negative  variation,  although 
we  now  often  express  it  by  the  newer  phrase,  current  of  action. 
The  change  briefly  is  that  any  excited  spot  becomes  momentarily 
electro-positive  to  other  parts  of  the  nerve,  and  this  is  propagated 
along  the  nerve  as  an  accompaniment  of  the  nervous  impulse. 

herefore,  we  place  two  non-polarisabje  electrodes,  f  and  d 


LECT.  VIIL]     GALVANOMETRIC  RESPONSE  OF  NERVE  103 


FIG.  ii. 


(Fig.  11),  a  few  millimeters  apart  upon  a  nerve,  one  end,  A,  of 
which  we  excite,  and  connect  the  electrodes  to  a  sensitive 
galvanometer,  immediately  the  impulse  reaches  the  situation  of 
the  first  electrode  /,  this  point  becomes  positive  to  d,  and, 
therefore,  a  current  flows  from  d  to  p  through  the  galvanometer. 
A  moment  later  the  two 
points  are  equipotential, 
and  no  current  flows ; 
a  minute  fraction  of  a 
second  later  this  balance 
is  upset,  that  is,  when 
the  impulse  reaches  the 
point  d ;  d  then  becomes 
positive  to  /,  and  so  the 
galvanometer  needle 
moves  in  the  opposite 
direction.  If  we  use 
the  electrometer,  the 

mercury  in  capillary  tube  moves  first  in  one  direction  then  in 
the  other.  Hence  the  expression  diphasic  variation.  If  instead 
of  single  stimuli  we  employ  a  series  of  shocks  as  during  fara- 
disation, the  galvanometric  reply  is  more  complex,  but  more 
distinctly  seen,  being  the  resultant  effect  of  the  individual 
stimuli. 

It  is  a  remarkable  fact  in  the  history  of  electro-physiology, 
how  our  knowledge  of  the  electrical  changes  in  nerves  has  been 
constructed  almost  altogether  from  experiments  on  the  nerves 
of  one  animal,  namely,  the  frog. 

v  '  Dr  Alcock,*  however,  has  filled  in  the  gap,  and  shown  that 
there  is  no  essential  difference  between  the  nerves  of  frogs, 
mammals,  and  birds  as  regards  their  negative  variation, 
excitability,  and  their  susceptibility  to  the  narcotic  effects  of 
anaesthetics. 

In   one   particular,  however,  there   is   a  difference,  and  one 

which   is  to  myself  most  interesting  ;    that  is  the  temperature 

necessary   to   extinguish    this    sign    of  life.       The    nerves    are 

paralysed    by   cold,   and    this    temperature    also   varies,   being 

*  P  roc.  Roy.  Soc.,  1903,  vol.  Ixxi.,  p.  264. 


io4  COAGULATION  TEMPERATURE  [LECT. 

—  3.5°  C.  in  the  frog,  —1.4°  in  the  hedgehog,  +3.8°  in  the 
rabbit,  and  +6.9°  in  the  pigeon.  The  influence  of  cold  is  not, 
however,  fatal ;  on  being  warmed  up  again  the  nerves  recover. 
The  differences  in  these  animals  in  the  cold  necessary  to  inhibit 
nervous  action  is  interesting  to  the  comparative  physiologist  in 
relation  to  the  normal  conditions  under  which  such  animals  live. 
The  cold-blooded  frog  requires  much  more  intense  cold  for  the 
purpose  than  the  warm-blooded  rabbit ;  and  the  hot-blooded 
pigeon  is  still  more  readily  affected  than  the  rabbit.  The 
hedgehog  is  an  animal  which  hibernates,  and  becomes  to  all 
intents  and  purposes  a  cold-blooded  animal  during  its  winter 
sleep.  We  see  it  occupies  an  intermediate  position  in  the 
figures  given,  between  the  frog  and  the  rabbit. 

Equally  interesting,  or  perhaps  even  more  so,  is  the  influence 
of  high  temperatures ;  the  negative  variation  is  abolished  at  40° 
in  frog's  nerve,  at  48°-49°  in  rabbit's  nerve,*  and  at  53°  C.  in 
pigeon's  nerve.  I  regard  this  fact  as  more  interesting  because 
we  are  able  to  explain  it.  The  extinction  point  corresponds 
with  the  first  coagulation  point  of  the  body  proteids,  and  thus 
heat  coagulation  is  the  cause  of  the  permanent  loss  of  irritability 
of  the  nerve.  You  will  remember,  we  have  seen  the  same  in 
muscle  ;  loss  of  irritability  is  produced  when  the  lowest  coagu- 
lating proteid  is  coagulated,  and  there  also  the  temperature 
varies  in  the  different  parts  of  the  animal  kingdom,  affording  an 
instance  of  what  we  then  called  biological  adaptation. 

Dr  Alcock  is  happily  here,  and  he  has  been  good  enough  to 
get  ready  a  typical  experiment.  I  expect  you  have  all  seen  a 
galvanometer  experiment  with  frog's  nerve,  so  he  has  selected 
a  rabbit's  nerve  for  his  demonstration.  We  will  have  the  lights 
lowered,  and  now  you  see  the  spot  of  light  reflected  on  to  the 
screen  from  the  galvanometer  mirror.  We  will  first  adjust  it  to 
the  zero  point,  and  throw  into  the  circuit  a  thousandth  part  of 
a  volt ;  you  see  the  spot  of  light  moves  along  the  screen  till  it 
reaches  division  number  25,  and  then  slowly  returns  to  zero. 

*  Eve,  in  work  on  the  cervical  ganglion  (Jour,  of  Phys.,  vol.  xxvi.,  p.  119), 
places  the  death  temperature  of  the  rabbit's  nerve-cells  at  the  same  point, 
46°  to  49°  C.,  and  Howell  made  corresponding  observations  on  the  tempera- 
ture necessary  to  abolish  the  conductivity  of  nerve  in  frogs  and  mammals. 


viii.]  HE  A  T  CONTRACTION  IN  NER  VE  105 

Now,  I  will  ask  Dr  Alcock  to  place  the  nerve  on  the  non- 
polarisable  electrodes,  and  to  excite  it  with  the  faradic  current 
for  a  few  seconds,  and  to  repeat  this,  always  exciting  for  the 
same  length  of  time,  first  while  the  nerve  is  at  30°,  secondly, 
when  he  has  raised  the  temperature  to  41°,  and  finally,  when 
the  temperature  is  49°  C,  and  to  write  his  results  upon  the 
blackboard. 

The  following  was  the  final  appearance  of  the  table  so 
constructed : — 

Temperature  of  Movement  of  spot  of  light 

the  Nerve.  in  divisions  of  scale. 

1.  30°  C.  9-5 

2.  30°  C.  9.5 

1.  41°  C.  10.0 

2.  41°  C.  10.6 

1.  49°  C.  o 

2.  49°  C.  o 

i/iooo  of  a  volt.  25 

You  see  quite  easily  how  a  temperature  of  41°  C.,  which  is 
sufficient  to  abolish  the  response  in  frog's  nerve,  has  a  little 
increased  the  vigour  of  a  mammalian  nerve ;  whereas  a  tem- 
perature of  49°  C.  has  killed  it. 

Heat  Contraction  in  Nerve 

It  is  possible  to  attack  the  same  problem  in  another  way. 
While  I  was  lecturing  to  you  on  heat  rigor  in  muscle,  and  Dr 
Brodie  was  showing  us  an  experiment  with  the  frog's  sartorius 
to  illustrate  his  method,  one  of  those  inspirations  which  so 
seldom  happen  to  a  speaker  in  the  course  of  a  lecture,  chanced 
to  occur  to  me.  It  was  this,  why  should  we  not  try  the  same 
experiment  with  nerve  ?  I  put  the  case  to  Dr  Brodie,  and  we 
have  subjected  the  question  to  the  test  of  experiment.  I  am 
now  able  to  tell  you  our  results,  and  show  you  a  typical 
experiment.*  At  first  we  were  disappointed  with  our  results ; 
the  shortening  in  a  nerve  was  very  small  and  often  absent ;  we 

*  At  the  University  of  London,  our  work  had  not  advanced  far  enough 
for  us  to  actually  show  this  experiment  ;  I  was,  however,  able  to  demonstrate 
it  successfully  at  New  York,  where,  in  consequence,  I  decided  to  omit  the 
galvanometer  experiment  of  Dr  Alcock, 


io6  COAGULATION  TEMPERATURE  [LECT. 

however,  obtained  better  results  with  the  spinal  cord.  We  soon 
found  that  the  reason  of  failure  was  that  we  employed  the  same 
apparatus  as  Brodie  used  for  muscle.  This  has  too  much  resist- 
ance for  a  nerve  to  move.  It  is  essential  that  the  parts  moved 
should  be  extremely  light,  and  free  from  friction.  This  we  have 
accomplished  by  the  very  simple  piece  of  apparatus  which  I 
have  on  the  table.  I  have  here  a  trough  containing  mercury 
covered  with  physiological  saline  solution  ;  a  frog's  sciatic  nerve 
lies  horizontally  in  the  salt  solution  on  the  surface  of  the  mer- 
cury;  it  is  fixed  at  one  end ;  the  free  end  is  attached  to  a  light 
aluminium  wire  suspended  vertically ;  at  the  upper  end  of  this 
is  a  little  mirror  which  reflects  a  spot  of  light  on  to  this  screen. 
The  trough  is  placed  in  a  water-bath,  and  carefully  warmed  ;  and 
as  the  nerve  shortens,  the  spot  of  light  moves  down  the  screen. 

The  temperature  is  now  36°  C,  and  the  light  has  shifted 
already  a  little,  but  it  is  not  until  we  reach  40°  that  the  move- 
ment becomes  energetic,  and  the  spot  moves  over  several  divisions 
of  the  scale.  This  is  exactly  the  same  as  in  muscle ;  the  small 
preliminary  movement  corresponds  to  what  in  a  proteid  solution 
we  should  term  the  stage  of  opalescence.  We  ought  properly 
to  keep  the  temperature  at  4O°-4i°  C.  for  at  about  half  an  hour, 
in  order  to  ensure  the  thorough  coagulation  of  all  of  this  first 
proteid  at  that  temperature.  The  actual  amount  of  this  proteid 
is  small,  as  judged  by  the  amount  of  shortening.  But  I  cannot 
expect  you  to  sit  patiently  here  and  watch  a  spot  of  light  for 
half  an  hour,  as  Brodie  and  I  do  in  our  dark  room.  So  we  will 
continue  the  heating,  and  now  at  47°  the  spot  once  more  starts 
moving,  and  at  48°-49°  has  walked  half-way  down  the  screen. 
Again,  you  must  imagine  a  half-hour  at  this  temperature  to 
elapse,  and  a  third  movement  is  then  visible  at  56°  to  58°  C. 

We  have  taken  the  rabbit  and  cat  as  instances  of  mammals ; 
the  proteid  in  nerve  coagulating  at  40°  C.  is  absent,  and  the  first 
proteid  produces  the  first  shortening  at  47°,  and  a  second  at  56°. 
There  is  also  a  third  and  very  pronounced  one  at  62°-63°,  which 
is  doubtless  due  to  the  connective-tissue  sheath  (see  coagulating 
point  of  tendon,  p.  54).* 

*  The  large  amount  of  connective  tissue  in  mammalian  and  birds'  nerves 
limits  useful  observation  beyond  that  temperature.  But  in  the  frog's  nerves^ 


VIIL]  HEAT  CONTRACTION  IN  NERVE  107 

Finally,  we  have  taken  the  pigeon  as  our  instance  of  a  hot- 
blooded  animal  (normal  temperature,  42°  C.),  and  here  we  find  the 
temperature  of  the  first  step  in  the  contraction  is  5O°-5  3°  C.  There 
is  a  second  at  58°-59°,  and  a  third  (connective  tissue)  at  63°  C. 

Heat  contraction  of  nerve  or  of  spinal  cord,  where  the  same 
facts  are  true,  thus  occurs  like  heat  rigor  in  muscle  in  successive' 
steps,  and  these  coincide  with  the  coagulation  temperatures  of 
the  proteids  contained  in  saline  extracts  of  nervous  tissues. 
The  ultimate  length  of  the  nerve  when  heat  contraction  is 
finished  is  usually  (in  the  frog)  about  half  its  original  length. 

The  method  is  capable  of  application  to  other  tissues ;  we 
have  already  made  some  experiments  with  strips  of  rabbits' 
liver,  and  the  shortenings  at  47°  and  56°  correspond  with  the 
coagulation  points  of  the  two  principal  proteids  found  in  saline 
extracts  of  that  organ.  In  frog's  liver  there  is,  as  in  muscle  and 
nerve,  an  extra  proteid  coagulating  at  36°-4O°  C.  Here,  how- 
ever, the  amount  of  shortening  is  less  than  in  structures  like 
muscle,  nerve,  and  tendon,  where  the  histological  elements  have 
a  longitudinal  direction. 

The  most  interesting  of  these  facts  from  a  comparative 
standpoint  is  the  coagulating  temperature  of  the  first  proteid  ; 
because  this  is  the  temperature  at  which  life  is  extinguished,  and 
the  electrical  response,  irritability,  and  conductivity  abolished. 

I  have  little  doubt  that  a  prolonged  exposure  to  tempera- 
tures a  few  degrees  below  those  given  by  Alcock  would  extin- 
guish the  electrical  response  in  nerve,  for  the  real  coagulation 
process  begins  and  occurs  slowly  then,  as  evidenced  by  slight 
shortening  in  experiments  on  heat  contraction,  and  by  opales- 
cence  in  experiments  with  saline  extracts. 

The  Coagulation   Temperature  of  Cell-Globulin ,  and  its  Bearing 
on  Hyperpyrexia 

Results  such  as  these  we  have  been  speaking  of  have  not 
only  an  academic  interest,  but  also  a  direct  practical  bearing  for 

the  contraction  due  to  the  connective  tissue  is  insignificant  in  amount ;  and 
on  heating  to  70°  a  further  shortening  occurs,  just  as  in  fractional  heat  coagu- 
lation of  saline  extracts  of  nervous  tissue  a  proteid  is  found  to  coagulate 
at  that  temperature, 


io8  COAGULATION  TEMPERATURE  [LECT. 

the  pathologist.  In  carrying  out  work  from  this  point  of  view,  I 
have  been  associated  with  Dr  Mott,  and  the  following  are  our 
results  and  conclusions. 

It  is  well  known  that  there  are  various  factors  that  influence 
the  temperature  of  heat  coagulation  of  proteid  substances. 
Among  these  the  rate  of  the  rise  of  temperature  is  one  of  some 
importance.  This  was  clearly  demonstrated  in  the  work  of 
Corin  and  Ansiaux,*  and  of  Hewlett,  f  These  observers  showed 
in  connection  with  serum  and  egg-white  respectively  that  if 
the  temperature  is  maintained  long  enough  below  the  point  at 
which  heat  coagulation  is  usually  stated  to  occur,  not  merely 
opalescence  but  the  formation  of  flocculi  will  take  place  (see 
also  p.  17). 

In  performing  the  process  of  fractional  heat  coagulation 
with  extracts  of  various  mammalian  organs  and  tissues,  I J 
have  shown  that  in  nearly  all  of  them  a  proteid  is  present 
that  coagulates  at  an  extremely  low  temperature,  which  varies 
in  different  cases  from  45°  to  50°  C.  This  proteid  is  a 
globulin,  and  has  been  variously  named.  Thus,  in  muscle 
we  have  learnt  to  call  it  para-myosinogen ;  in  liver  cells  it 
has  been  called  hepato  -  globulin  ;  in  extracts  of  nervous 
tissues,  neuro-globulin  ;  in  extracts  of  lymph-cells,  cell-globulin, 
and  so  on.  There  can  be  very  little  doubt  that  such  a 
globulin  is  characteristic  of  protoplasmic  structures,  and  even 
if  it  is  not  absolutely  the  same  proteid  in  all  cases,  the 
term  cell-globulin  may  be  provisionally  employed  in  a 
general  sense  to  indicate  that  cells,  as  a  rule,  yield  to  saline 
solvents  a  proteid  with  characteristically  low  coagulation 
temperature. 

One  might,  however,  object  that  the  behaviour  of  saline 
extracts  of  cells  does  not  necessarily  teach  us  the  condition 
of  the  proteids  as  they  are  actually  present  in  the  complex  we 
call  protoplasm.  In  view  of  such  a  criticism,  I  attach  special 
importance  to  the  researches  subsequently  carried  out  by  Brodie 

*  Bulletin  de  Pacad.  roy.  de  Belgique,  1891,  vol.  xxi.,  p.  3. 
t  Jour,  of  Phys.^  1893,  vol.  xiii.,  p.  494. 

\  See  Schafer's  Text-Book  of  Physiology ',   vol.  i.,  art.  "The  Chemistry 
Of  the  Tissues  and  Organs,"  by  W.  D.  Halliburton. 


viii.  HYPERPYREXIA  109 

and  Richardson,*  and  later  by  Vernon.f  We  have  already 
seen  that  these  investigations  show  in  the  case  of  muscle  that 
the  shortening  which  occurs  in  the  process  of  heat  rigor  is  not 
a  single  one,  but  takes  place  in  a  series  of  steps  ;  the  tempera- 
tures at  which  these  steps  occur  are  the  same  as  those  at  which 
the  individual  proteids  separate  out  during  the  fractional  heat 
coagulation  of  an  extract  of  muscular  tissue.  Thus  in  mammalian 
muscle  the  two  principal  shortenings  occur  at  47°  and  56°  C, 
the  coagulation  temperatures  of  the  two  principal  muscular 
proteids.  In  frog's  muscle  there  are  three  steps  at  40°,  47°,  and 
56°  C.  respectively,  which  correspond  to  the  three  proteids  that 
can  be  separated  out  in  a  saline  extract  of  this  variety  of  muscular 
tissue. 

I  may  also  remind  you  once  more  that  Brodie  and  Richardson 
showed  another  important  point,  namely,  that  after  the  first 
step  has  occurred  in  the  shortening,  the  muscles  lose  their 
irritability ;  in  other  words,  in  order  to  destroy  the  vitality  of 
muscular  tissue,  it  is  not  necessary  to  raise  the  temperature 
sufficiently  high  to  coagulate  all  its  proteids,  but  that  when  one 
of  the  muscular  proteids  has  been  coagulated,  the  living 
substance  as  such  is  destroyed.  It  therefore  appears  to  be  the 
case  that  the  proteids  of  muscle  are  not  independent  units. 
The  unit  is  protoplasm,  and  if  one  of  its  essential  constituents 
is  destroyed,  protoplasm  as  such  ceases  to  exist. 

These  experiments  in  connection  with  muscle  would  lead 
one  to  suppose  that  the  same  is  true  in  regard  to  other  proto- 
plasmic structures ;  that  is  to  say,  the  results  which  have  been 
obtained  by  the  examination  of  saline  extracts  of  such  structures 
can  be  applied  to  the  elucidation  of  the  composition  of  the 
protoplasm  of  which  they  are  composed. 

This  last  sentence,  which  is  word  for  word  what  I  wrote  a 
few  years  ago,  has  been  abundantly  justified  by  the  experiments 
with  nerve,  which  have  formed  the  subject  of  the  first  two 
sections  of  this  lecture. 

*  Phil.  Trans.,  1899,  vol.  cxci.  B.,  p.  127. 
t  Jour,  of  Phys.,  1899,  vol.  xxiv.,  p.  239. 


I  id  COAGULATION  TEMPERATURE  [LECT. 

Dr  Mott's  attention  was  directed  to  a  consideration  of  this 
subject  in  connection  with  the  question  of  hyperpyrexia.  He 
found  that  the  nerve-cells  after  death  from  this  condition  show 
a  diffuse  staining  with  methylene  blue,  and  a  disappearance  or 
breakdown  of  the  Nissl  granules. 

It  is  a  familiar  fact  that  very  high  body  temperature  is 
incompatible  with  life.  Marinesco  *  has  pointed  out,  in  experi- 
ments on  hyperthermia  in  mammals,  that  a  temperature  of  47°  C. 
is  immediately  fatal;  a  temperature  of  45°  C.  kills  in  an  hour 
or  two  ;  a  temperature  of  43°  C.  kills  after  a  longer  lapse  of 
time.  Moreover,  the  occurrence  of  death  is  coincident  with  the 
breakdown  of  the  nerve-cells  in  the  manner  just  indicated.  It 
is  probable  that  analogous  changes  occur  in  other  cells  of  the 
body  also,  but  these  have  not  yet  been  specially  investigated. 
The  nerve-cells  are  undoubtedly  essential  to  healthy  life,  and 
lend  themselves  very  readily  to  microscopic  investigation, 
especially  by  the  methylene-blue  process.  A  temperature  of 
47°  C.  leads  to  an  instantaneous  disappearance  of  the  chromato- 
phile  granules ;  the  same  change  occurs  at  45°  C.  in  a  few 
hours ;  at  43°  C.  a  longer  lapse  of  time  is  necessary. 

We  were  struck  with  the  coincidence  of  the  fatal  temperature 
(47°  C.)  with  that  of  the  coagulation  temperature  of  neuro- 
globulin ;  and  we  argue  that,  as  in  muscle,  the  coagulation  of 
even  the  lowest  coagulating  proteid  of  nerve-cells  would  produce 
a  destruction  of  the  life  of  their  protoplasm  ;  a  distinct  chemico- 
physical  explanation  can  therefore  be  found  for  death  due  to 
hyperpyrexia.^ 

Still  a  temperature  as  high  as  47°  C.  (117°  F.)  in  man  is 
unknown  ;  and  we  thought  it  possible  that  the  proteid  in  question 
would  coagulate  at  a  lower  temperature  if  it  was  kept  at  that 

*  "  Recherches  sur  les  Lesions  des  Centres  Nerveux  consecutives  a 
1'Hyperthermie  Experimentale  et  a  la  Fievre,"  Revue  Neurologique,  1899. 
See  also  Goldscheider  and  Flatau,  Norm.  u.  path.  Anat.  d.  Nervenzellen, 
Berlin,  1898. 

t  In  cold-blooded  animals,  a  far  lower  temperature  is  fatal,  which  is 
quite  intelligible,  seeing  their  tissues  contain  a  proteid  which  coagulates 
below  40°  C. 


VIIL]  HYPERPYREX1A  m 

temperature  a  sufficient  length  of  time.  We  proceeded  to  put 
the  suggestion  to  the  test  of  experiment,  fully  anticipating,  in 
the  light  of  the  work  of  Hewlett  and  others,  alluded  to  (p.  108), 
that  the  supposition  would  turn  out  to  be  correct.  Experiment 
has  shown  that  this  is  the  case. 

The  first  experiments  were  made  with  the  brains  of  cats. 
After  the  animal  had  been  killed  by  bleeding  (sufficient  chloro- 
form having  been  given  to  render  it  unconscious),  the  brain  was 
rapidly  removed  ;  the  grey  matter  was  finely  minced  and  ground 
up  in  a  mortar  with  0.9  per  cent,  solution  of  sodium  chloride. 
We  selected  this  solvent  as  the  one  likely  to  produce  least 
change  in  the  constituents  of  the  protoplasm.  After  repeated 
filtration,  the  extract  remained  somewhat  opalescent ;  it  was 
fairly  rich  in  proteid,  as  tested  by  rapidly  boiling  a  sample.  It 
did  not  prove  at  all  difficult  to  see  any  increase  in  the  opal- 
escence  when  the  extract  was  carefully  heated  in  a  water-bath. 
The  extract  was  faintly  alkaline,  but  we  judged  it  best  not  to 
add  any  acid  to  neutralise  this,  in  order  that  we  might  deal  with 
as  natural  conditions  as  possible. 

When  the  rate  of  observation  is  fairly  rapid,  the  first  crop  of 
flocculi  was  observed  to  separate  out  at  47°  C.  These  are 
removable  by  filtration,  and  the  filtrate  is  clear. 

In  our  next  experiment  the  temperature  was  not  allowed  to 
rise  higher  than  45°,  and  was  kept  between  44°  and  45°  C.,  being 
more  frequently  nearer  the  lower  than  the  higher  of  these  limits. 
In  somewhat  less  than  two  hours  the  separation  of  flocculi 
took  place,  and  as  good  a  coagulum  was  ultimately  obtained 
at  this  temperature  as  was  obtained  in  the  first  experiment 
at  47°  C.  Previous  to  the  formation  of  actual  flocculi,  there 
was  an  increase  of  opalescence,  which  became  denser  as  time 
went  on. 

In  the  next  experiment  an  attempt  was  made  to  obtain  the 
coagulum  at  a  still  lower  temperature,  namely,  42°  C.  (108°  F.) ; 
here  again  we  were  rewarded  with  success ;  there  was  at  first 
the  gradual  deepening  of  the  opalescence,  and  in  time  a  distinct 
separation  of  minute  flocculi,  which  increased  in  number  and 


ii2  COAGULATION  TEMPERATURE  [LECT. 

size.  The  first  separation  of  visible  flocculi  occurred  about 
three  hours  after  the  commencement  of  the  observation,  and  an 
hour  later  the  crop  was  fairly  abundant,  though  the  size  of  the 
coagulum  was  not  so  great  as  in  the  previous  two  experiments. 
After  filtering,  the  flocculi  were,  of  course,  removed,  but  the 
filtrate  was  still  distinctly  opalescent.  Doubtless,  if  we  had  con- 
tinued to  watch  the  tube  for  a  longer  time  the  coagulation  would 
have  been  more  complete. 

The  next  experiment  consisted  in  trying  a  still  lower 
temperature,  namely,  4O°-4i°  C.  In  this  case,  however,  though 
the  tube  was  watched  for  eight  hours,  there  was  no  coagula- 
tion. 

We  have  repeated  this  series  of  experiments  several  times, 
and  in  some  cases,  instead  of  grinding  up  the  brain  substance 
with  salt  solution  only,  we  have  employed  clean  sand  or 
powdered  glass  as  well.  By  this  means  one  obtains  an  extract 
richer  inproteid,  filtration  is  easier,  and  the  filtrate  clearer.  The 
phenomena  of  heat  coagulation  are  exactly  the  same  as  in  the 
experiments  just  described,  but  theproteid  being  more  abundant? 
they  are  more  readily  seen. 

In  a  further  series  of  experiments  we  have  employed  human 
grey  matter,  removed  from  the  cadaver  as  soon  as  possible  after 
death.  We  have  selected  the  optic  thalamus  as  a  convenient 
mass  of  grey  matter  for  this  purpose.  The  results  absolutely 
agree  with  those  already  given. 

We  had  hoped  to  have  had  an  opportunity  of  similarly  investi- 
gating the  grey  matter  after  death  had  supervened  in  hyper- 
pyrexia ;  but  since  we  began  this  work  no  such  case  came  under 
our  notice.  We  have  accordingly  had  to  be  content  with 
experiments  on  animals.  A  cat  was,  after  anaesthetisation, 
rapidly  killed  by  bleeding ;  the  brain  was  removed  as  quickly  as 
possible,  and  divided  into  two  equal  halves ;  this  was  first  done 
roughly,  and  then  the  two  halves  were  accurately  made  equal 
by  removing  fragments  of  the  white  matter  from  the  heavier 
moiety.  Each  weighed  about  9.5  grammes.  One  half  was 
immediately  ground  up  with  powdered  glass  and  normal  saline 


VIIL]  HYPERPYREXIA  113 

solution,  and  the  extract  examined.  The  other  half  was  first 
heated  to  47°  C.  for  an  hour,  and  then  similarly  treated,  the 
same  volume  of  saline  solution  being  used.  In  the  extract  of 
the  first  (the  normal)  half,  fractional  heat  coagulation  revealed 
the  presence  of  coagula,  which  came  down  at  47°,  56°-6o°,  and 
72°  C.  respectively.  In  the  extract  of  the  second  (the  heated) 
.half,  the  47°  coagulum  was  absent,  but  the  other  two  were 
obtained.  The  total  -amount  of  proteid  in  the  two  extracts 
was  also  estimated  in  the  usual  way,  by  weighing  the  pre- 
cipitate produced  by  excess  of  alcohol.  100  c.c.  of  the  first 
(normal)  extract  contained  0.674  grammes ;  100  c.c.  of  the 
second  (heated)  extract  contained  only  0.144  grammes  of 
proteid  material.  The  amount  of  cell-globulin  which  passes 
into  solution  in  normal  saline  is  thus  relatively  large. 

In  a  second  experiment,  the  half -brain  was  heated  to 
42°  C.  instead  of  47°  C.  It  was  kept  at  42°  C.  for  five  hours. 
Examination  of  the  extracts  showed  that  the  extract  of  the 
normal  half  gave  the  usual  crop  of  coagula,  and  100  c.c.  con- 
tained 0.483  grammes  of  proteid ;  the  extract  of  the  half-brain 
which  had  been  heated  to  42°  C.  gave  as  before  no  coagulum 
at  47°  C. ;  100  c.c.  of  this  extract  contained  0.226  grammes 
of  proteid.  The  chemical  examination  of  brain  tissue  as 
fresh  as  possible  thus  gave  results  which  exactly  correspond  to 
those  obtained  in  the  experiments  with  saline  extracts  of  brain. 

The  same  is  true  for  the  histological  examination  we  have 
made  with  "  surviving  "  brain  tissue. 

We  have  not  repeated  Marinesco's  experiments  on  hyper- 
thermia  in  a'nimals,  but  we  have  performed  the  experiment  of 
exposing  the  brain  in  situ  immediately  after  death  to  an 
elevated  temperature.  Two  cats  were  anaesthetised  and 
decapitated  ;  the  heads  were  placed  in  a  warm  chamber,  a 
thermometer  being  inserted  into  the  brain  through  the  foramen 
magnum.  In  one  case  the  brain  was  kept  at  44°  to  45°  C.  for 
one  and  a  half  hours ;  in  the  second,  at  42°  to  43°  for  three 
and  a  half  hours.  In  each  case,  and  particularly  in  the  first 
one,  the  cells  exhibited  chromatolysis. 

H 


ii4  COAGULATION  TEMPERATURE  [LECT. 

The  accompanying  photo-micrograph  (Fig.  12)  shows  the 
appearance  of  the  cells  from  a  case  of  hyperpyrexia  in  a 
boy. 

We  may,  therefore,  sum  up  by  saying  that  our  experiments 
confirm  our  hypothesis,  that  the  physico-chemical  cause  of 
death  from  hyperpyrexia  is  due  to  the  coagulation  of  cell- 
globulin.  When  this  constituent  of  cell-protoplasm  is  coagulated . 
the  protoplasm  as  such  is  destroyed.  The  temperature  at 
which  such  coagulation  is  most  easily  produced  is  47°  C.  But 
temperatures  as  low  as  42°  C.  will  have  the  same  effect,  provided 
the  heating  is  continued  long  enough.  These  chemical  changes 
in  the  brain  substance  are  demonstrable  by  experiments  with 
saline  extracts  of  that  tissue,  or  with  the  "  surviving  "  brain  of 
animals  just  killed.  They  are  coincident  with  the  histological 
(chromatolytic)  changes  in  nerve-cells,  which  can  be  rendered 
evident  by  the  use  of  the  methylene-blue  method.  The 
-^expression  coagulation  necrosis  employed  by  Marinesco  for  this 
appearance  is,  therefore,  justifiable,  though  Marinesco  and  other 
histologists  who  have  obtained  similar  results  missed  the  connec- 
tion of  the  temperature  necessary  to  produce  it,  with  that  of  the 
coagulation  temperature  of  cell-globulin.  Lastly,  though  the 
nerve-cells  are  those  which  lend  themselves  most  readily  to  the 
histological  part  of  the  research,  it  is  by  no  means  improbable 
(looking  at  the  wide  distribution  of  cell-globulin),  that  many 
other  cells  of  the  body  are  affected  by  a  high  temperature  in  a 
corresponding  manner. 

It  will  be  noticed  throughout  this  part  of  our  work,  we 
were  concerned  with  mammals  only,  because  our  object  was 
to  draw  conclusions  on  the  subject  of  hyperpyrexia  in  man. 
As  was  previously  pointed  out  (p.  no,  footnote),  it  is  perfectly 
well  known  that  cold-blooded  animals,  like  frogs,  are  killed 
by  a  lower  temperature  than  mammals,  and  this  depends  on 
the  presence  of  the  additional  proteid  in  their  tissues,  which 
coagulates  at  36°-4O°  C.  In  the  process  of  evolution,  when 
animals  were  formed  higher  in  the  scale  which  we  term  warm- 
blooded, and  which  maintain  a  normal  temperature  as  high 


FlG.  12. — Section  of  spinal  cord  from  a  case  of  hyperpyrexia,  the  temperature  being 
109°  F.  before  death.  The  whole  of  the  cells  throughout  the  central  nervous 
system  showed  a  diffuse  homogeneous  staining  with  methylene  blue.  The  Nissl 
granules  had  entirely  disappeared  from  the  processes  and  body  of  the  cells,  and 
the  stainable  substance  had  a  fine,  dust-like  appearance.  Magnification,  400 
diameters. — MOTT. 


[To  face  page  114. 


VIIL]  HYPERPYREXIA  115 

as  the  death  temperature  of  a  frog,  the  cells  adapted  themselves 
to  the  altered  circumstances,  and  this  proteid  of  their  proto- 
plasm disappeared.  Again,  in  birds,  with  a  still  higher  normal 
temperature,  there  is  a  further  adaptation,  as  pointed  out  on 
pp  104-107. 


LECTURE    IX 

THE   CHEMICAL   PATHOLOGY  OF   CERTAIN    DEGENERATIVE 
NERVOUS   DISEASES 

I  PROPOSE  in  the  present  lecture  to  deal  with  more  strictly 
pathological  questions.  The  new  subject  of  chemical  pathology 
has  a  great  future  before  it,  and  this  is  as  true  for  nervous 
diseases  as  for  diseases  of  other  parts  of  the  body.  Up  to 
the  present  time  comparatively  few  pathologists  have  worked 
at  the  chemical  side  of  nervous  disease ;  and  a  few  years  ago, 
when  Mott  and  I  started  our  work,  this  branch  of  investigation 
was  almost  untouched. 

Chemical  Pathology  of  General  Paralysis  of  the  Insane 

The  first  disease  which  attracted  our  attention  is  the  very 
common  one  known  as  General  Paralysis  of  the  Insane ;  and  I 
will  first  deal  with  our  results  in  that  disease,  because  it  is  a 
typical  example  of  a  degenerative  disease,  in  which  there  is 
considerable  destruction  of  nervous  tissue.'  It  is  a  para-syphilitic 
affection  like  tabes,  with  which  it  is,  pathologically  speaking, 
identical.  It  is  a  premature,  primary,  progressive  decay  of  the 
neuron,  affecting  especially  those  structures  which  have  been 
developed  latest.  To  the  naked  eye  the  extensive,  degenerative, 
and  wasting  process  which  occurs,  especially  in  the  frontal  and 
central  convolutions,  is  perfectly  evident.  Microscopical  examina- 
tion of  the  diseased  brains  reveals  degenerative  changes 
in  the  cells ;  and  the  perivascular  lymphatics  are  seen  (by 
Marchi's  method  of  staining)  in  acute  cases  to  contain 

phagocytes  filled  with  black-stained  fatty  matter.     During  the 
nc 


LECT.  ix.]      GENERAL  PARALYSIS  OF  THE  INSANE  iij 

course  of  the  disease  there  are  seizures  of  an  epileptiform  or 
apoplectiform  kind,  and  after  recovery  of  the  patient  from 
each  of  these  fits  he  is,  as  a  rule,  worse  mentally.  Each  fit 
indicates  the  breakdown  of  a  new  focus  of  cerebral  matter. 

The  place  of  the  atrophied  brain  substance  within  the 
cranium  is  taken  by  excess  of  cerebro-spinal  fluid.  It  is  often 
possible  to  obtain  as  much  as  100  to  200  c.c. 

The  main  object  of  our  research  was  to  examine  the  cerebro- 
spinal  fluid,  and  to  attempt  to  discover  in  it  some  substance  or 
substances  derived  from  the  disintegration  of  the  brain-matter, 
which,  passing  thence  into  the  general  circulation,  would  produce 
auto-intoxication,  and  thus  account  for  some  of  the  symptoms 
of  the  disease. 

Normal  cerebro-spinal  fluid  is  alkaline  to  litmus,  and  contains 
a  very  small  percentage  of  solids  (see  first  Lecture).  The  fluid 
from  cases  of  General  Paralysis  is  not  only  more  abundant,  but 
is  also  richer  in  solids ;  this  is  principally  due  to  excess  of 
proteid  material.  The  average  percentage  of  proteid  in  eight 
specimens  was  0.24,  that  is  about  three  times  as  much  as  is 
found  in  cerebro-spinal  fluid  in  cases  of  spina  bifida.  It  is 
alkaline,  like  the  normal  fluid.  Fibrinogen  is  absent,  as  in  the 
normal  fluid ;  proteoses  and  peptone  are  also  absent.  There  is 
a  small  quantity  of  albumin  present ;  in  the  normal  fluid 
albumin  is  absent.  The  most  abundant  proteid,  however,  is 
nucleo-proteid  ;  in  one  case  sufficient  was  present  to  produce- 
intravascular  coagulation,  when  10  c.c.  of  the  fluid  were  injected 
into  the  jugular  vein  of  a  cat. 

Another  distinction  between  the  normal  and  the  pathological 
fluid  is  the  presence  in  the  former  of  a  reducing  substance,  and 
the  absence  of  this,  as  a  rule,  in  the  latter ;  in  only  two  out  of 
fourteen  cases  was  it  found.* 

But  the  most  noteworthy  distinction  between  the  two  fluids 
is  the  presence  of  abundance  of  choline  in  the  specimens 
removed  from  cases  of  General  Paralysis.  The  chemical  examina- 
tion of  the  fluid  supports  our  contention  that  the  cerebro-spinal 
fluid  acts  as  the  lymph  of  the  brain,  and  when  the  disintegration 

*  This  may  possibly  have  been  due  to  glycolysis,  as  all  these  fluids  were 
removed  from  the  cadaver.  • 


ti8  DEGENERATIVE  NERVOUS  DISEASES  [LEG*. 

of  the  cerebral  tissue  is  increased,  as  in  General  Paralysis,  the 
fluid  contains  the  products  of  such  disintegration  (e.g.,  choline, 
nucleo-proteid).  The  greater  number  of  the  specimens  of 
cerebro-spinal  fluid  we  examined  were  removed  from  the  cadaver 
as  soon  as  possible  after  death.  We  also  had  the  opportunity 
of  examining  four  specimens  removed  during  life  by  lumbar 
puncture  from  cases  of  General  Paralysis.*  The  results  obtained 
with  these  are  identical  with  those  obtained  from  the  post-mortem 
specimens.  We  have  also  secured  on  several  occasions  blood 
removed  for  remedial  purposes  from  such  patients  by  vene- 
section, and  we  regard  one  of  the  most  important  outcomes  of 
our  work,  the  discovery  that  the  blood  also  contains  the  same 
—  toxic  material  during  a  seizure. 

Let  me  now  trace  the  way  in  which  we  were  led  to  identify 
the  substance  in  cerebro-spinal  fluid  as  choline. 

We  found  that  the  cerebro-spinal  fluid  itself  when  injected 
into  the  blood  stream  of  a  living  anaesthetised  animal  (rabbit, 
cat,  or  dog)  produced  a  fall  of  blood  pressure,  whereas  normal 
cerebro-spinal  fluid  has  no  such  effect.  We  at  first  thought 
it  was  the  proteid  material  in  the  fluid  which  was  responsible 
for  this  result.  But  we  found  that  the  removal  of  the  proteid 
by  heat  or  by  alcohol  makes  no  difference  in  the  observed 
result.  Our  next  supposition  was  that  the  active  substance 
might  be  inorganic.  We  therefore  took  a  large  quantity  of 
the  fluid,  evaporated  it  to  dryness,  and  incinerated  the  residue. 
The  ash  was  dissolved  in  physiological  salt  solution,  and  injected 
with  a  negative  result.  The  active  substance  is  therefore  of 
organic  nature,  but  is  not  proteid.  We  then  took  the  alcoholic 
filtrate  of  the  fluid,  evaporated  off  the  alcohol  at  40°  C,  took 
up  the  solid  residue  with  absolute  alcohol,  filtered  it,  and  again 
evaporated  off  the  alcohol.  This  was  repeated  two  or  three 
times  more,  in  order  to  lessen  the  danger  of  any  potassium 
salts  remaining  in  solution.  The  final  residue  was  dissolved 
in  physiological  saline  solution  ;  we  injected  this,  and  obtained 
a  fall  of  pressure  like  that  produced  by  the  original  fluid.  The 
active  substance  is  therefore  of  organic  nature,  and  one  which 

*  We  were  indebted  for  these  to  Dr  John  Turner  of  the  Essex  County 
Asylum. 


IX.]  ACTION  OF  C HO  LINE  AND  NEURINE  119 

is  soluble  both  in  alcohol  and  in  physiological  saline  solution. 
It  then  occurred  to  us  that  the  substance  might  be  alkaloidal; ', 
we  accordingly  took  a  solution  of  it  in  saline  solution"~prepared 
as  just  described,  and  added  to  it  phospho-tungstic  acid  in  the 
presence  of  sulphuric  acid  until  no  more  precipitate  occurred. 
We  then  took  both  precipitate  and  filtrate,  and  from  the  former 
separated  out  a  base  which  produced  a  fall  of  blood  pressure  ; 
whilst  from  the  latter  (the  filtrate)  no  such  material  was 
obtainable.  We  naturally  next  thought  of  the  bases  which 
would  most  likely  be  present ;  the  base  which  we  first  thought 
of  was  choline.  It  accordingly  became  necessary  to  make  an 
examination  of  the  physiological  action  of  pure  choline,  to 
which  we  added  a  corresponding  examination  of  the  action 
of  the  closely  related  base  neurine. 

At  the  same  time,  it  was  necessary  for  us  to  identify  choline 
and  neurine  by  means  of  chemical  tests. 

We  will  first  take  the  physiological  action  of  the  two  bases, 
and  then  their  chemical  reactions.  We  may  anticipate  the 
result  by  saying  that  the  substance  in  cerebro-spinal  fluid 
proved  to  be  identical  in  its  action,  and  in  its  chemical 
behaviour,  with  choline.  Neurine  is  not  present. 

Physiological  Action  of  Choline  and  Neurine 

We  injected  quite  small  quantities  (i  to  5  c.c.  of  a  0.2  per 
cent  solution  either  of  choline  itself  or  of  its  hydrochloride),  for 
we  sought  as  far  as  possible  to  note  the  effects  produced  by 
solutions  of  such  strength  as  would  be  comparable  to  the 
amount  of  the  base  presumably  present  in  pathological  cerebro- 
spinal  fluid.  Its  actions  are  briefly  as  follows  : — 

(1)  Choline    produces   a   temporary   fall    in   arterial   blood- 
pressure. 

(2)  This  is  in  some  measure  due  to  its  action  on  the  heart. 

(3)  It  is  also,  and  probably  mainly,  due  to  dilatation  of  the 
peripheral  vessels,  especially  in  the  intestinal  area. 

(4)  The  kidney  and  limbs   undergo  a  passive  lessening   of 
volume  secondary  to  the  fall  in  general  arterial  pressure. 

(5)  The  spleen  contracts  markedly,  and  when  this  passes  off, 
its  normal  curves  are  greatly  exaggerated. 


120 


NERVOUS  DISEASES 


[LECT. 


(6)  We   obtained  no  evidence  of  any  direct  action  of  the 
base  on  the  cerebral  vessels. 

(7)  The  action  on  the  splanchnic  vessels  is  due  to  the  direct 
action  of  the  drug  on  the  neuro-muscular  apparatus  of  those 


FlG.  13. — Original  size.  Tracing  of  intestinal  oncometer  (/.#.)  and  arterial  blood 
pressure  (B.P^  in  a  cat ;  10  c.c.  of  cerebro-spinal  fluid  from  a  case  of  General 
Paralysis  were  injected  ;  the  same  effect  was  obtained  in  the  same  animal  by 
injecting  2  c.c.  of  a  0.2  per  cent,  solution  of  choline.  The  Ml  of  blood  pressure 
is  at  first  mainly  cardiac  in  origin,  for  the  oncometer  tracing  first  follows  the 
fall  of  arteri.il  blood  pressure  passively ;  it,  however,  soon  rises,  indicating 
dilatation  of  the  peripheral  vessels. 

vessels,  for  after  the  influence  of  the  central  nervous  system 
has  been  removed  by  section  of  the  spinal  cord,  or  of  the 
splanchnic  nerves,  choline  still  causes  the  typical  fall  of  arterial 


ix.]  ACTION  OF  CHOLINE  AND  NEURIN&  tit 

pressure.  The  action  of  peripheral  ganglia  was  in  other 
experiments  excluded  by  previously  poisoning  the  animal  with 
nicotine. 

(8)  Choline   has    very   little    effect    on    the   galvanometric 
response    of    nerve-trunks ;    neurine   has   a    depressing    effect 
(Waller  and  Sowton). 

(9)  There  is  no  effect  on  the  respiration. 

(10)  Section    of    the   vagi    makes    no   difference    in    these 
experimental  results. 

(11)  Previous   atropinisation  of  the   animal  causes  a  great 
difference  ;  it  abolishes  the  fall  of  arterial  pressure,  though  there 
is  still  some  dilatation  of  splanchnic  blood-vessels.     In  fact,  very 
often  injection  of  choline  after  atropine  produces  a  rise  instead 
of  a  fall  of  blood  pressure. 

This  last  point  has  already  been  illustrated  by  the  tracings 
in  Figs.  4  and  5  (p.  84). 

In  all  these  particulars  choline  and  the  basic  substance 
separated  out  from  the  cerebro-spinal  fluid  or  blood  of  these 
patients  are  in  complete  agreement.  How  close  the  agreement 
is  can  be  seen  by  the  numerous  tracings  reproduced  in  our 
Royal  Society  paper. 

At  the  actual  lecture  many  of  these  tracings  were  shown  as 
lantern  slides.  I  will  be  content  here  with  giving  one  very 
typical  tracing  (Fig.  13),  which  shows  the  fall  of  pressure,  and 
the  accompanying  vaso-dilatation  as  recorded  by  Edmund's 
intestinal  oncometer. 

It  is  obviously  impossible  with  any  one  specimen  to  perform 
all  the  physiological  tests  enumerated  under  the  foregoing 
eleven  heads.  The  most  readily  available  test,  and  which  I  shall 
subsequently  speak  of  as  the  physiological  test,  is  the  fall  of 
blood  pressure  with  accompanying  intestinal  vascular  engorge- 
ment. The  animal  next  receives  a  small  dose  of  atropine,  and 
a  further  injection  of  choline  then  produces  either  no  fall  or  it 
may  be  a  rise  of  blood  pressure  instead. 

This  important  test  was  demonstrated  on  an  anaesthetised  dog  at  the 
lecture. 

It  is  unnecessary  to  enter  into  full  details  of  the  physiological 


122 


DEGENERATIVE  NERVOUS  DISEASES 


[LECT. 


action  of  neurine,  as  it  was  never  found  in  the  cerebro-spinal 
fluid  or  blood   of  these  patients.     Suffice  it  to  say  that   it  is 


much  more  poisonous,  it  affects  the  heart  more  (and  the  effect 
on  the  heart  is  neutralised  by  atropine),  causes  peripheral 
constriction  rather  than  dilatation  of  the  peripheral  vessels, 


ixj      CHEMICAL  TESTS  FOR  CttOUtfE  AND  NEURINE      123 

stimulates  and  then  paralyses  the  respiratory  movements,  and 
has  a  curare-like  action  on  the  voluntary  muscles. 

It  will  be  sufficient  to  reproduce  one  tracing  of  the  action 
of  neurine.  I  have  selected  one  which  shows  the  effect  on 
arterial  pressure  and  respiration  (Fig.  14).  I  have  inserted  it 
to  show  that  there  can  never  be  any  confusion  in  the  physio- 
logical test  between  choline  and  neurine. 

Chemical  Reactions  of  Choline  and  Neurine 

The  repeated  treatment  with  absolute  alcohol  alluded  to  in 
the  method  we  adopted  of  separating  out  the  base  from  the 
cerebro-spinal  fluid  and  blood  of  these  patients,  had  for  its 
object  the  removal  of  proteid,  and  of  salts  of  ammonium  and 
potassium.  In  order  to  ensure  as  little  admixture  of  these 
inorganic  salts  as  possible,  it  is  essential  that  the  alcohol  used 
should  be  as  water-free  as  possible.  The  final  residue  was 
crystalline  at  first,  but  soon  deliquesced  on  exposure  to  air. 
It  is  soluble  in  water,  physiological  saline  solution,  alcohol,  and 
ether.  On  putrefaction  it  gives  off  the  odour  of  ammonia  and 
trimethylamine.  Dissolved  in  water  or  in  physiological  salt 
solution,  it  gives  white  precipitates  with  phospho-tungstic  acid, 
phospho-molybdic  acid,  and  mercuric  chloride.  It  gives  a 
brownish  precipitate  with  iodine  dissolved  in  a  solution  of 
potassium  iodide  or  in  alcohol,  a  yellow  precipitate  with  gold 
chloride,  and  with  platinum  chloride.  The  alcoholic  solution 
gives  with  gold  chloride  a  precipitate  which  consists  of  tiny 
yellow  crystals  which  are  soluble  in  hot  water  and  hot  alcohol ; 
they  are  insoluble  in  ether. 

In  all  these  points  the  base  from  the  cerebro-spinal  fluid  or 
blood  exactly  resembles  choline. 

The  aqueous  solution  gives  no  precipitate  with  tannin, 
which  distinguishes  it  from  neurine.  The  two  bases  are  also 
readily  distinguishable  by  means  of  their  chromates,  that  of 
neurine  being  scarcely  soluble,  while  that  of  choline  is  readily 
soluble  in  cold  water  (Cramer  *). 

*  Jour,  of  Phys.,  1904,  vol.  xxxi.,  p.  30.     Cramer  also  found  no  neurine 
from  the  decomposition  of  protagon. 


124 


DEGENERATIVE  NERVOUS  DISEASES 


[LECT. 


In  my  experience,  the  most  readily  applicable  and  the  most 
delicate  of  these  chemical  tests  is  the  platinum  test*  An 
alcoholic  solution  of  platinum  chloride  is  added  to  an  alcoholic 
solution  of  choline  hydrochloride.  The  precipitated  platino- 
chloride  is  of  a  yellow  colour,  easily  soluble  in  water,  and  so 
is  distinguishable  from  the  platino-chloride  of  ammonium  and 
of  potassium.  It  is  insoluble  in  ether,  and  readily  soluble  in 
1 5  per  cent,  alcohol. 

On  evaporating  the  solution  in  15  per  cent,  alcohol  to  dry- 


FlG.  15. — Crystals  of  the   platino-chloride   of  choline  prepared   from  a   solution   of 
choline  hydrochloride.     Crystallised  from  15  per  cent,  alcohol. 

ness  at  a  low  temperature  (40°  C),  it  crystallises  out  in  yellow 
octahedra,  shown  in  the  accompanying  drawings. 

It  is  always  dangerous  to  attempt  to  identify  any  substance 

merely   by    its    crystalline    form.     It    is    well    known    that    the 

platino-chlorides  of  potassium,  of  ammonium,  and  of  bile  salts 

crystallise  in  a  very  similar  way.     So  in  cases  where  there  was 

*  See  also  Gulewitsch,  Zeit.f,  physiol.  Chem.,  vol.  xv.,  p.  149. 


IX.] 


CHEMICAL  TESTS  FOR  CHOLINE 


125 


sufficient  material  to  work  with,  we  further  examined  the  char- 
acters of  the  choline  platino-chloride,  and  found  that  its  solu- 
bilities, the  amount  of  platinum  it  contains,  and  the  fact  that  on 
heating  it  gives  off  trimethylamine,  are  sufficient  to  definitely 
distinguish  it  from  the  others  mentioned. 

When  the  platino-chloride  of  choline  is  dissolved  in  water 
and  allowed  to  crystallise,  the  crystals  formed  are   six-sided 


FIG,  1 6. — Crystals  of  the  platino-chloride  of  the  base  separated  from  cerebro-spinal 
fluid  in  cases  of  General  Paralysis  of  the  Insane.  Crystallised  from  15  per  cent, 
alcohol.  The  identity  of  this  base  with  choline  is  not  only  shown  by  the  form 
of  these  crystals,  but  by  its  solubilities  and  by  other  numerous  reactions  described 
in  the  text. 

plates  and  needles  of  a  yellow  colour.  These  usually  form 
curious  aggregations  described  by  Donath  *  as  star-fish  crystals, 
with  four  rays,  one  usually  much  larger  than  the  others.  These 
crystals  are  regarded  by  Donath  as  more  typical  than  the 
octahedra  Mott  and  I  figured,  and  form  an  even  better  micro- 
scopic chemical  test  for  small  amounts  of  choline. 

*  Zeit.f.  physiol.  Chem.,  1903,  vol.  xxxix.,  p.  526. 


126  DEGENERATIVE  NERVOUS  DISEASES  [LECT. 

I  may  sum  up  the  main  results  which  we  obtained  in  cases 
of  General  Paralysis  in  the  following  way. 

The  cerebro-spinal  fluid  of  patients  suffering  from  the  disease 
contains  toxic  material,  which  originates  from  the  disintegration 
of  nervous  tissues  mainly  in  the  brain.  We  obtained  chemical 
evidence  of  the  existence  of  nucleo-proteid  in  the  fluid,  and 
though  the  amount  of  nucleo-proteid  is  not,  as  a  rule,  sufficient 
to  cause  massive  intravascular  clotting  when  the  fluid  is  injected 
into  animals,  we  consider  that  the  presence  of  even  small 
quantities  continuously  being  poured  out  into  the  cerebro-spinal 
fluid,  collecting  in  the  perivascular  lymphatics  and  passing 
thence  to  the  blood,  will  produce  harmful  results.  The  idea 
suggested  itself  to  our  minds  that  an  increase  in  the  coagula- 
bility of  the  blood  in  the  small  vessels  of  the  cerebral  region, 
which  nucleo-proteid  would  produce,  might  form  a  determining 
factor  in  promoting  venous  stasis,  and  thus  are  caused  the  acute 
manifestations  or  seizures  of  apoplectiform  or  epileptoid  nature 
which  the  patients  exhibit. 

The  other  toxic  substance  which  we  succeeded  in  isolating 
is  choline.  The  existence  of  choline  in  the  cerebro-spinal  fluid 
and  also  in  the  blood,  is  a  clear  indication  of  the  disintegration 
of  the  brain  tissue.  Choline,  however,  is  not  a  very  poisonous 
substance.  Glycero-phosphoric  acid  and  lactic  acid  are  even 
less  toxic. 

Still,  even  choline  has  some  effect.  A  feeble  circulation  and 
fatty  degeneration  of  the  heart  are  very  frequent  concomitants 
of  the  terminal  stages  of  the  disease,  especially  after  a  series  of 
seizures,  and  the  idea  seems  feasible  that  choline  may  in  part 
explain  these.  A  single  dose  of  choline  in  a  dog  or  cat 
produces  but  little  effect  on  the  heart ;  still  there  is  some  effect, 
and  it  does  not  appear  a  far-fetched  idea  to  suppose  that  the 
continual  pouring  of  small  doses  of  choline  into  the  cardiac 
^tissue  may  in  time  produce  cardiac  weakness,  and  even 
degeneration. 

After  prolonged  convulsions,  the  blood  pressure  of  these 
patients  (as  tested  by  the  Hill-Barnard  sphygmometer)  falls 
considerably ;  it  rises  again  a  few  days  after  the  convulsions 
have  ceased.  There  can  be  no  doubt  that  the  convulsions  are 


ix.]  CHOL1NE  IN  CEREBRO-SP1NAL  FLUID  127 

associated  with  the  breaking  down  of  nervous  tissue,  and  we 
therefore  think  it  probable  that  the  choline  so  liberated  is 
responsible  for  the  fall  of  pressure  which  occurs  then. 

Whether  choline  will  explain  the  fits  themselves,  is  another 
question.  We  were  never  successful  in  producing  anything 
like  a  fit  in  our  experiments  on  anaesthetised  animals,  even 
when  we  injected  quite  strong  doses  of  the  base  into  the  carotid 
artery.  Donath,  however,  has  stated  that  by  injecting  the 
material  into  the  cerebral  substance  in  the  region  of  the  sensori- 
motor  convolutions,  he  has  produced  convulsive  attacks  in 
animals.  If  this  is  confirmed  it  is  an  indication  that  choline  is  a 
more  toxic  material  than  Mott  and  I  considered  it  to  be. 


Cholinetn  the  Blood,  and  Cerebro- Spinal  Fluid  in  other 
Degenerative  Diseases  of  the  Nervous  System 

We  have  now  seen  that  in  the  disease  called  General 
Paralysis  of  the  Insane,  the  degenerative  changes  that  occur  in 
the  central  nervous  system  are  associated  with  the  presence  of 
the  products  of  such  degeneration  in  the  cerebro-spinal  fluid. 
One  of  these  products,  choline,  is  derived  from  the  breakdown 
of  lecithin.  Choline  can  be  identified  in  the  blood  also  of  these 
patients.  The  tests  on  which  one  relies  for  the  detection  of  this 
alkaloid  are  mainly  two  :  the  first  is  a  chemical  test,  namely, 
the  obtaining  of  the  typical  yellow  crystals  of  the  platino- 
chloride  from  the  alcoholic  extract  of  the  blood.  These  crystals 
have  not  only  a  definite  form,  but  their  solubilities  distinguish 
them  from  other  somewhat  similar  crystals,  as  also  does  the  fact 
that  they  yield  a  fixed  percentage  of  platinum,  and  give  rise  to 
an  odour  of  trimethylamine  when  decomposed  by  heat.  The 
second  test  is  a  physiological  one :  a  saline  solution  of  choline, 
of  choline  hydrochloride,  and  of  the  residue  obtained  from  the 
alcoholic  extract  of  the  cerebro-spinal  fluid  and  blood  of  these 
patients,  produce  a  temporary  fall  of  pressure  when  injected 
intravenously  in  animals.  This  fall  is  partly  cardiac  in  origin, 
and  partly  due  to  dilatation  of  peripheral  blood-vessels  ;  the 
dilatation  is  due  to  the  direct  action  of  the  alkaloid  on  the 
neuro-muscular  mechanism  of  the  blood-vessels  themselves. 


128  DEGENERATIVE  NERVOUS  DISEASES  [LECT. 

There  are  many  substances  which  produce  a  fall  of  arterial 
pressure,  but  choline  is  peculiar  in  the  fact  that  after  the 
administration  of  a  small  dose  of  atropine,  it  no  longer  produces 
a  fall  but  a  rise  of  blood  pressure,  or,  at  any  rate,  the  fall  is 
abolished. 

In  the  investigation  of  the  blood,  as  a  rule,  only  a  small 
amount  of  material  has  been  at  one's  disposal,  and  in  order  to 
obtain  satisfactory  evidence  of  choline  it  is  necessary  to 
considerably  concentrate  the  alcoholic  extract.  It  is  therefore 
necessary  to  limit  the  number  of  tests  to  be  performed.  The 
two  tests,  however,  appear  to  be,  if  positive,  quite  conclusive 
evidence  of  the  presence  of  choline. 

The  only  other  organic  substance  with  which  I  am 
acquainted  which  gives  the  physiological  test  is  spermine,* 
but  the  chemical  reactions  of  this  substance  are  so  different 
from  those  of  choline  that  there  can  be  no  risk  of  confusing 
the  two. 

Bile  salts,  which  Croftan  f  has  shown  to  be  present  in  blood, 
give  a  platino-chloride  of  similar  crystalline  form  and  colour  to 
that  given  by  choline  when  the  crystallisation  takes  place  from 
15  per  cent,  alcohol;  but  when  crystals  are  obtained  by  the 
evaporation  of  an  aqueous  extract,  as  in  Donath's  method,  the 
peculiar  star-fish  crystals  so  typical  of  the  choline  compound  are 
never  obtained  (O.  Griinbaum  J).  In  the  matter  of  the 
physiological  test,  solutions  of  bile  salts  do  produce  a  slight  fall 
of  arterial  pressure ;  but  quite  strong  solutions  (5  per  cent.)  are 
necessary  to  produce  the  result,  and  this  effect  is  not  abolished 
by  atropinisation. 

Solutions  of  potassium  chloride  and  of  ammonium  chloride 
produce  a  fall  of  blood  pressure,  but  again  this  is  not  abolished 
by  atropine.  The  similarity  of  the  platino-chlorides  has  been 
already  alluded  to.  Contamination  with  these  inorganic  sub- 
stances may  be  largely  excluded  by  the  use  of  absolutely 
water-free  alcohol,  and  if  they  are  present  the  choline  platino- 
chloride  can  be  separated  out  by  dissolving  it  in  water,  and 

*  Dixon,  Jour,  of  Phys.,  vol.  xxv.,  p.  356. 

t  American  Jour,  of  Med.  Science ',  1902,  p.  150. 

t  Private  communication  to  the  author. 


IX.] 


CHOL1NE  IN  THE  BLOOD 


129 


recrystallising  ;   it  then  shows  the  star-fish  crystals  previously 
alluded  to.* 

The  difficulty  of  obtaining  cerebro-spinal  fluid  during  life  led 
Mott  and  myself  in  our  later  work  to  devote  more  particular 
attention  to  the  blood  ;  unfortunately,  choline  does  not  pass  into 
the  urine,  so  the  withdrawal  of  a  small  quantity  of  blood  from 
the  patient  is  necessary.  Ten  c.c.  of  blood 
will  give  the  tests  in  a  marked  case.  This 
is  a  point  of  some  practical  importance  ; 
in  cases  where  it  is  difficult  to  distinguish 
between  serious  cases  of  organic  disease 
and  cases  of  so-called  functional  neurosis, 
the  performance  of  the  tests  described 
may  come  to  the  assistance  of  the  practical 
physician  in  making  his  diagnosis. 

Normal  blood  used  in  these  quantities 
gives  negative  results.  If  the  platino- 
chloride  is  crystallised  from  alcohol,  a 
certain  number  of  octahedra  may  be  found  ; 
these  may  be  due  to  small  quantities  of 
choline,  but  are  more  probably  due  to  con- 
tamination with  potassium  and  ammonium 
chlorides.  The  same  is  true  for  ordinary 
dropsical  effusions. 

General  Paralysis  is  not  the  only  disease 
where  there  is  disintegration  of  nervous 
tissues  ;  it  was,  therefore,  to  be  anticipated 
that  choline  would  be  discoverable  in  the 
blood  in  other  nervous  diseases,  and  we 

identified  it  in  many.     Some  of  these  are  diseases  of  the  central 
nervous  system  (disseminated  sclerosis,  combined  sclerosis,  etc.)  ; 

*  The  want  of  care  in  the  use  of  absolute  alcohol  will  probably  explain 
most  of  the  criticisms  on  the  methods  which  have  been  made  by  Vincent 
and  Cramer  (Jour,  of  Phys.,  vol.  xxx ,  p.  143),  and  by  Allen  and  French 
(Proc.  Phys.  Soc.,  1903,  p.  xxix  ;  Jour,  of  Phys.,  vol.  xxx.).  More  lately,  Allen 
has  found  that  the  iodine  test  for  choline  can  be  employed  without  any  danger 
of  confusion  with  inorganic  chlorides.  By  means  of  this  test,  he  has  con- 
firmed our  statements  on  the  presence  of  choline  in  the  blood  of  patients 
suffering  from  extensive  degeneration  in  nervous  tissue  (ibid.,  xxxi.,  p.  Ivi.) 


FlG.  17. — Nerve  degene- 
ration in  alcoholic  neu- 
ritis ;  stained  with  osmic 
acid  (S.  MARTIN). 


130  DEGENERATIVE  NERVOUS  DISEASES  [LECT. 

some  of  the  peripheral  nervous  system  (Beri-beri  and  other 
forms  of  neuritis).  The  degeneration  of  peripheral  nerves  in 
alcoholic  neuritis  is  shown  in  the  accompanying  drawing  (Fig.  17). 


FlG.  1 8. — The  uppermost  line  represents  the  respiration,  taken  by  the  tambour 
method.  The  next  line  is  the  blood  pressure  from  the  carotid  of  a  cat.  The 
next  is  a  time-tracing  in  seconds  ;  the  next  is  the  signal  line,  the  raising  of  which 
indicates  the  period  of  the  injection.  The  lowest  line  is  the  abscissa  of  the  blood 
pressure. 

The  injection  produced  as  usual  no  effect  on  respiration.  The  actual  volume  of  saline 
solution  of  the  active  material  was  5  c.c.  This  was  injected  into  the  external 
jugular  vein. 

FlG.  1 8  represents  the  fall  of  arterial  pressure  produced  by  the  injection  into  the 
external  jugular  vein  of  the  choline  obtained  from  10  c.c.  of  the  blood  in  a  case 
of  Beri-beri.  This  tracing  is  of  the  original  size. 

We   found    that    the    two  tests    fitted    together  with  great 
accuracy.     If  the   chemical    test    is   performed   first,   one   can 


ix.]  TESTS  FOR  CHOL1NE  IN  BLOOD  131 

prophesy,  from  the  amount  of  crystals,  the  result  of  the  injec- 
tion. If,  on  the  other  hand,  the  physiological  test  is  performed 
first,  one  can  prophesy  accurately  from  the  fall  of  blood  pressure 
whether  an  abundant  or  scanty  crop  of  crystals  will  be  obtained. 
The  graphic  records  we  have  of  these  physiological  experi- 
ments are  very  numerous,  but  the  two  given  in  Figs.  18  and  19  will 
serve  as  a  typical  sample.  The  case  selected  is  one  of  Beri-beri. 


FlG.  19. — The  result  of  injecting  the  same  volume  of  the  same  solution  in  the  same 
cat  after  atropine  had  been  administered.     There  is  now  a  rise  of  blood  pressure. 

Since  our  joint  work  was  finished  on  this  subject,  Dr  Mott 
has  made  further  investigations  on  the  value  of  the  choline  test 
(i.e.  the  chemical  test),  for  active  degeneration  of  the  nervous 
system,*  and  concludes  that  the  test  is  of  no  use  to  decide 
whether  a  case  is  organic  or  functional  unless  the  organic 
disease  is  active  at  the  time  the  blood  is  drawn  ;  it  is  therefore 
specially  applicable  after  the  onset  of  new  symptoms  indicative 
of  irritative  or  destructive  processes. 

Since  our  papers  were  published  several  other  investigators 
have  applied  themselves  to  the  same  problem,  and  in  spite  of 
*  Archives  of  Neurology,  vol.  ii.,  p.  858. 


132  DEGENERATIVE  NERVOUS  DISEASES  [LECT. 

the  criticisms  previously  alluded  to,  the  main  results  confirm 
our  original  statements. 

Gumprecht*  examined  cerebro-spinal  fluid,  and  found 
minute  traces  of  choline  in  the  normal  condition.  This  is 
enormously  increased,  not  only  in  General  Paralysis  but  in  many 
other  diseases  of  the  nervous  system.  He  directed  his  attention 
in  particular  to  acute  diseases  like  meningitis. 

Donathf  also  worked  mainly  with  cerebro-spinal  fluid,  but 
made  also  a  few  observations  on  the  blood.  The  cerebro-spinal 
fluid  was  obtained  by  lumbar  puncture  during  life.  He 
introduced  some  new  details  into  the  method  adopted,  so  as  to 
make  the  test  more  certain.  He  invariably  finds  choline  in 
organic  nervous  diseases,  and  gives  a  long  list  of  the  cases  he 
examined.  It  is  absent  in  functional  diseases.  It  is  interesting 
to  note  that  in  one  of  the  cases  of  neurasthenia  he  examined, 
the  test  was  positive.  This  suggests  either  an  incorrect 
diagnosis  ;  or,  if  the  diagnosis  was  correct,  neurasthenia  is  not 
always  a  purely  functional  disorder. 

Dana  and  Hastings  J  have  examined  the  cerebro-spinal 
fluid  of  a  large  number  of  cases  mainly  from  the  point  of  view 
of  cyto-diagnosis,  but  in  two  cases  of  alcoholic  psychosis,  where 
they  instituted  also  a  search  for  choline,  they  found  it. 

Otto  Griinbaum  has,  more  recently  still,  investigated  the 
question,  and,  as  this  has  been  done  at  a  time  after  the  appearance 
of  the  criticisms  of  Allen  and  French,  and  of  the  suggestions  of 
Donath,  it  may  be  regarded  as  the  most  satisfactory  series  of 
experiments  yet  made.  His  results  with  the  chemical  test 
applied  to  blood  are  as  follow  : — 

In  normal  blood,  choline  is  absent  or  present  only  in 
negligible  quantities. 

In  four  cases  of  herpes  zoster,  where  degenerative  changes 
are  slight,  the  result  was  uniformly  negative.  The  degenerative 
change  must,  therefore,  be  fairly  extensive  to  get  a  positive 
result.  In  four  cases  of  hysteria,  and  one  case  of  tobacco  poison- 
ing, the  result  was  also  negative. 

*  VerhandL  der  Congr.  fur  i?inere  Med.^  Wiesbaden,  1900,  p.  326. 
t  Zeit.f.physioL  Chem.y  1903,  vol.  xxxix.,  p.  526. 
}  Medical  Record^  New  York,  23rd  January  1904. 


ix.]  TESTS  FOR  CHOLINE  IN  BLOOD  133 

He  obtained  positive  results  in  three  cases  of  disseminated 
sclerosis,  in  two  of  paralysis  agitans,  in  three  of  tabes  dorsalis,  in 
one  of  progressive  muscular  atrophy,  in  two  of  transverse 
myelitis,  and  one  of  myasthenia  gravis. 

In  one  case  of  progressive  muscular  atrophy,  in  one  of 
disseminated  sclerosis,  and  in  one  of  transverse  myelitis,  he 
obtained  negative  results ;  but  this  really  does  not  militate 
against  the  general  conclusion,  for  in  all  three  cases  the  disease 
had  reached  a  quiescent  stage. 

In  a  second  paper  just  published  by  Donath,*  he  has 
extended  his  work  on  the  cerebro-spinal  fluid,  and  shown  that 
the  amount  of  phosphoric  acid  in  it  is  increased  also  when  the 
degenerative  lesion  is  sufficiently  great  This  is  what  one 
would  expect,  for  phosphoric  acid,  like  choline,  is  a  product  of 
lecithin  decomposition.  In  functional  conditions  like  epilepsy, 
melancholia,  and  hysteria,  this  increase  of  phosphoric  acid  does 
not  occur. 

Lecithin  is  a  substance  which  should  be  also  interesting  to  the  pathologist 
from  quite  a  different  point  of  view.  Preston  Kyes  t  has  advanced  the  view 
that  it  may  play  the  part  of  the  complement  in  toxin  poisoning,  e.g.  in  the 
case  of  snake  venom. 

*  Zeit.f.physiol.  Chern.,  1904,  vol.  xlii.,  p.  141.  In  the  same  number  of 
this  journal  (p.  157),  G.  Mansfield  criticises  Donath's  researches  upon 
choline.  I  have  no  doubt  he  will  be  able  to  satisfactorily  dispose  of  these 
criticisms. 

t  Ibid.,  vol.  xli.,  p.  273. 


LECTURE  X 

DEGENERATION  AND  REGENERATION  OF  NERVES 

IF  one  takes  any  animal  cell,  such  as  an  amoeba,  and  divides  it 
into  two  parts,  one  part  possessing  the  nucleus,  and  the  other 
not,  the  former  continues  to  live  and  thrive,  the  latter 
degenerates  and  dies.  This  general  truth  concerning  the 
importance  of  the  nucleus  in  regulating  the  nutrition  of  the  cell 
receives  special  importance  when  applied  to  the  units  of  the 
nervous  system,  because  the  fact  that  axons  degenerate  when 
cut  off  from  the  bodies  of  the  cells  of  which  they  are  outgrowths, 
has  furnished  physiologists  with  one  of  their  most  valuable 
methods  of  tracing  and  differentiating  tracts  of  nerve-fibres. 
The  degeneration  of  the  distal  segment  of  the  nerve-fibre  is  a 
rapid  and  marked  change,  known  after  its  discoverer  as 
Wallerian  degeneration.  The  slower  atrophic  changes  in  the 
proximal  segment  which  take  place  if  regeneration  does  not 
occur,  are  known  as  disuse  atrophy ;  under  these  circumstances, 
the  chromatolytic  changes  in  the  protoplasmic  body  of  the  cell 
is  evidence  of  the  relative  inactivity  of  the  cell-body. 

The  microscopic  appearance  of  degenerated  nerve-fibres  is 
well  known,  and  Mott  and  myself,  approaching  the  subject  from 
the  chemical  point  of  view,  have  sought  to  compare  and 
correlate  the  chemical  changes  that  occur  with  these  histological 
alterations  of  structure. 

For  this  purpose,  we  took  a  series  of  cats,  and  divided  both 
sciatic  nerves  in  the  upper  part  of  the  thigh.  The  animals  were 
killed  at  varying  intervals  after  this  operation,  their  blood  was 
collected,  and  the  nerves  themselves  examined  both  histo- 
logically  and  chemically.  The  histological  method  principally 

134 


LECT.  x.]  CHOLINE  IN  BLOOD  AFTER  SECTION  OF  NERVES  135 

employed  was  the  Marchi  reaction  ;  our  main  object  of  examin- 
ing the  blood  was  to  ascertain  whether  choline  was  present,  and 
the  macro-chemical  methods  in  the  examination  of  the  nerves 
consisted  chiefly  in  ascertaining  the  proportion  of  water  and 
solids,  and  the  amount  of  phosphorus  in  the  solids. 


Experiments   witJi   the   Blood 

These  may  be  very  briefly  stated  ;  the  methods  used  were 
the  same  as  those  already  described  in  connection  with  human 
blood. 

The  blood  of  normal  cats  contains  the  merest  traces  of 
choline.  A  few  crystals  can 
generally  be  found  by  the 
platinum  test,  though  it  is 
quite  possible,  in  view  of 
recent  criticisms,  that  some 
or  all  of  these  crystals  may 
have  been  due  to  a  slight 
contamination  with  chlo- 
rides of  potassium  or  am- 
monium. The  amount  of 
these  crystals  was  too 
small  to  admit  of  a  more 
detailed  examination  of 
them.  If  choline  is  pres- 
ent, it  occurs  in  too  small 
an  amount  to  give  the 
physiological  test  when  20 
to  30  c.c.  of  the  blood  are 
employed. 

When  signs  of  degene- 
ration set  in,  evidence  of 
chemical  breakdown  of 
lecithin  was  found  in  the 
presence  of  choline  in  the 

blood ;    it  was  detectable  three   or  four  days  after  the  opera- 
tion of  cutting  the  nerves  had  been  performed.     The  best  yield 


FlG.  20. — Result  of  injecting  choline  obtained 
from  30  c.c.  of  blood  of  a  cat  eight  days  after 
section  of  both  nerves. 


1 36  DEC EN £R A  TlON  AND  REGENERA  TION          [LEG*. 

of  crystals,  and  the  most  marked  fall  of  blood  pressure  was 
in  the  case  of  the  eight-day  cat.  By  this  time  the  Marchi 
method  showed  pronounced  histological  degeneration.  This 
fall  was  abolished  by  atropine  (see  Figs.  20  and  21). 

In  the  blood  of  the  cat  killed 
thirteen  days  after  the  operation, 
there  was  a  good  positive  result, 
but  there  was  less  choline  present ; 
from  this  time  onward  the  evidence 
of  choline  steadily  diminished,  until 
the  normal  was  reached  in  the  later 
stages  of  the  degenerative  process. 

Chemical  Examination  of  the 

Nerves 

The  nerves  were  carefully  dis- 
sected out,  weighed,  dried  to  con- 
stant weight  at  iio°  C,  and  again 
weighed.  The  dry  residue  was 
used  for  the  determination  of 
phosphorus.  Considering  the  small 
weight  of  the  nerves,  we  judged 
this  would  give  more  accurate  re- 
sults than  any  attempt  to  isolate 

fr  IG.  21. — Result  of  injection  of  the  11 

same  amount  after  atropine.  tne  fatty  material,  and  determine 

the  phosphorus  in  that. 

The  dried  nerve  was  soaked  in  5  per  cent,  hydrochloric  acid 
for  three  weeks,  in  order  to  get  rid  of  inorganic  phosphates. 
Such  treatment  apparently  gets  rid  of  the  phosphorus  combined 
as  nuclein  or  nucleo-proteid  ;  for  in  many  of  the  nerves  of 
later  date  there  was  considerable  nuclear  proliferation  seen 
microscopically,  and  yet  little  or  no  phosphorus  was  obtained 
from  the  nerves  after  treatment  in  this  way  with  hydrochloric 
acid.  The  phosphorus  we  did  obtain  came,  therefore,  either 
wholly  or  chiefly  from  the  phosphorised  fat.  The  nerves  were 
then  dissolved  on  the  water-bath  at  '100°  C.  in  fuming  nitric 
and  sulphuric  acids,  to  which  an  occasional  pinch  of  potassium 


x.]         CHEMISTRY  OF  WALLERIAN  DEGENERATION        13? 

chlorate  was  added.  The  heating  with  acid  was  continued  for 
many  hours.  The  phosphate  so  formed  was  precipitated  by 
ammonium  nitro-molybdate.  The  yellow  precipitate  so  obtained 
was  washed,  and  dissolved  in  dilute  ammonia,  then  precipitated 
by  magnesia  mixture ;  this  precipitate  was  incinerated  and 
weighed  as  magnesium  pyrophosphate,*  from  which  the  amount 
of  phosphorus  was  calculated. 

The  following  table  gives  the  main  results  ;  particulars  are 
added  relating  to  the  choline  in  the  blood,  and  the  histological 
condition  of  the  nerves. 


Days  after 
section. 

Cat's  Sciatic  Nerves. 

Condition  of 
Blood. 

Condition  of 
Nerves. 

Water. 

Solids. 

Percentage  of 
Phosphorus 
in  Solids. 

Normal     . 
1-3  ... 
4-6  .     .     . 

65.I 
64.5 
69o 

34-9 
35-5 
30.7 

I.I 

0.9 

0.9 

(Minimal    traces 
of        choline 
present. 
Choline      more 

f  Nerves  irritable 
and  histologi- 
{     cally  healthy. 
Irritability  lost  ; 

abundant. 

degeneration 

beginning. 

8    ... 

10     ... 

13    ... 

68.2 
70.7 
71-3 

31.8 
29-3 

28.7 

0-5 
o.3 

0.2 

1  Choline     abun- 
f     dant. 

^Degeneration 
I      well      shown 
~|      by  Marchi  re- 
^     action. 

{Marchi  reaction 

25-27    .    • 
29    ... 

72.1 
72-5 

27.9 
27.5 

traces 

0.0 

f  Choline     much 
\     less. 

still  seen,  but 
absorption  of 
degenerated 

fat  has  set  in. 

44-60  .     . 

72.6 

27.4 

0.0 

Choline  almost 

Absorption      of 

100-106    . 

66.2 

33-8 

0.9 

disappeared. 
Choline  almost 

fat  complete. 
Return  of  func- 

disappeared. 

tion  ;    nerves 

regenerated. 

We  see  from  the  foregoing  table  that  the  nerves  remained 
excitable  up  to  the  third  day,  and  were  chemically  and  histo- 
logically  healthy.  Beyond  this  date  early  signs  of  degeneration 
set  in ;  the  amount  of  phosphorised  material  in  the  nerves 
slightly  dropped,  and  the  amount  of  choline  in  the  blood  slightly 

*  A  full  description  of  this  method  of  phosphorus  estimation  is  given  in 
the/^/r.  of  Phys.,  1892,  vol.  xiii.,  pp.  814,  821. 


1 38  DEGENERA  TION  AND  REG  EN  ERA  T10N          [LECT  . 

increased.  On  or  about  the  eighth  day,  the  Marchi  reaction 
became  strongly  marked.  This  date  is  coincident  with  a 
great  drop  in  the  amount  of  phosphorus  in  the  nerves,  and 
with  the  appearance  of  a  large  quantity  of  choline  in  the 
blood. 

The  Marchi  reaction  remained  at  its  acme  up  to  the 
thirteenth  day,  and  the  amount  of  phosphorus  in  the  nerves 
became  less  and  less.  The  amount  of  choline  in  the  blood 
was  lessened ;  it  therefore  appears  that  of  the  disintegration 
-products  of  lecithin,  the  choline  is  earliest  removed ;  the  phos- 
phorus probably  in  the  form  of  phosphoric  acid  next,  and 
the  non-phosphorised  fat  which  remains  gives  the  black 
colour  with  Marchi's  reagent.  This  fat,  however,  is  absorbed 
in  time. 

By  the  twenty-seventh  day,  the  phosphorus  had  nearly,  and 
by  the  twenty-ninth  day  entirely,  disappeared.  The  removal 
of  the  fat  had  also  commenced,  so  that  the  particles  which  stain 
black  with  Marchi's  fluid  were  less  numerous. 

At  the  forty-fourth  day,  the  removal  of  the  fat  was  all  but 
complete,  and  little  remained  except  shrunken  empty  nerve 
tubules  and  connective  tissue.  This  date  is,  however,  variable, 
for  the  condition  was  not  so  far  advanced  in  another  cat  sixty 
days  after  the  nerves  had  been  cut.  At  any  rate,  in  comparison 
with  the  central  nervous  system,  the  date  of  entire  removal  of 
fatty  particles  is  an  early  one. 

Regeneration  begins  about  the  same  time ;  that  is,  about 
the  sixtieth  day  in  nerves  which  had  united  spontaneously,  and 
a  little  earlier  in  cases  where  the  loose  ends  of  the  nerves  had 
been  sutured  together. 

By  the  hundredth  to  the  hundred-and-sixth  day  regeneration 
was  well  marked,  especially  in  sensory  fibres,  and  the  nerves 
were  once  more  excitable.  The  fibres  were  seen  to  be  fine,  and 
many  were  medullated ;  they  took  stains  normally.  Their 
chemical  condition  had  also  almost  returned  to  the  normal. 
The  first  sign  of  the  return  of  phosphorus  was  seen  with  the 
commencement  of  myelination  on  the  sixtieth  day,  but  it  was 
well  marked  on  the  hundredth  day. 

In  normal  nerves  the  percentage  of  phosphorus  is  a  little 


Xj  CHEMICAL  AND  HISTOLOGICAL  CHANGES  139 

over  I  per  cent.  In  the  regenerated  nerves  analysed,  it  was  a 
little  under  I  per  cent. 

Whether  all  the  phosphorus  in  the  regenerated  fibres  was 
in  the  medullary  sheath,  or  partly  in  the  comparatively  large 
axis-cylinder,  it  ts  impossible  to  say. 

With  regard  to  the  amount  of  water  in  the  nerves,  the  table 
shows  that  it  increases  with  the  degeneration,  and  continues 
high  while  absorption  is  occurring.  It  sinks  to  the  normal 
when  regeneration  has  set  in.  The  degenerated  nerves  show 
to  the  microscope  a  loose  texture  and  enlargement  of  the  lymph 
spaces  which  will  account,  in  part  at  any  rate,  for  the  increase 
of  water. 

Histological  Examination  of  the  Nerves 

Here  I  have  very  little  that  is  new  to  present  to  you,  though 
it  may  interest  you  to  see  reproductions  of  photo-micrographs 
of  our  preparations.*  The  general  result  of  the  microscopic 
study  of  the  nerves  will  have  been  gathered  from  the  foregoing 
summary  of  our  main  conclusions.^ 

I  may,  however,  remind  you  that  in  Wallerian  degeneration, 
changes  occur  in  all  three  parts  of  a  nerve-fibre ;  the  most 
prominent  changes  are  those  seen  in  the  medullary  sheath, 
which  undergoes  fragmentation  into  irregular  droplets  of 
myelin.  It  is  owing  to  this  circumstance  that  most  of  the 
staining  reactions  depend  upon  which  we  rely  to  detect 
degenerated  nerve  -  fibres.  The  axis  -  cylinder  undergoes  a 
corresponding  break-up,  and  upon  this  depends  the  loss  of 
function  which  occurs.  The  primitive  sheath  or  neurilemma 
undergoes  a  change  also.  Its  nuclei  multiply ;  we  noted  this 
in  our  preparations  made  from  the  eight-day  cat,  but  not 
before  that  date.  In  the  central  nervous  system,  where  the 
fibres  have  no  primitive  sheath,  there  is  naturally  no  multi- 
plication of  its  nuclei,  but  an  overgrowth  of  neuroglia  occurs 
instead.  It  is  possible  that  the  overgrowth  of  neurilemmal 
cells  in  the  one  case,  and  of  neuroglia  in  the  other,  may 

*  Shown  at  the  lecture  as  lantern  slides. 

t  Full  protocols  are  given  in  our  original  paper  in  the  Phil.  Trans. 


1 40  bEGENERA  TION  AND  REGENERA  TlON        [LECT  . 

be  the  result  of  irritation  set  up  by  the  products  of  chemical 
disintegration.      V 

We  have  made  no  special  study  of  the  central  ends  of  the  divided  nerves  ; 
this,  however,  has  been  done  by  Noll.*  He  performed  some  of  his  work 
on  large  animals  (horses),  and  so  could  obtain  sufficient  material  from  the 
central  end,  both  for  histological  purposes  and  for  chemical  analysis. 
Corresponding  to  what  is  termed  "  disuse  atrophy,"  he  found  some  dimin- 
ution in  the  amount  of  phosphorised  fat  in  this  region,  but  the  lessening 
is  not  so  marked  as  in  the  peripheral  portion  of  the  nerve.  He  puts  the 
date  of  disappearance  of  the  phosphorised  fat  in  the  peripheral  end  of  the 
nerve  at  28  days  ;  this  and  many  other  of  his  facts  fit  in  very  well  with 
our  work. 

Before  passing  on  to  the  study  of  the  specimens,  I  want 
to  detain  you  first  by  a  description  of  the  Marchi  method. 

The  Marchi  reaction  consists  in  placing  small  pieces  of 
nervous  tissue  in  Marchi's  fluid  (a  mixture  of  osmic  acid  and 
Muller's  fluid)  after  previous  hardening  in  M tiller's  fluid.  Under 
these  circumstances  healthy  nerve-fibres  are  stained  a  greenish- 
grey  colour,  but  degenerated  nerve-fibres  are  stained  an  intense 
black.  In  the  later  stages  of  degeneration,  when  the  fatty 
products  of  the  decomposition  of  the  fibres  have  been  absorbed, 
this  black  staining  is  naturally  no  longer  observable.  It  is 
important,  also,  to  observe  that  ordinary  neutral  fats,  such  as 
are  contained  in  adipose  tissue,  give  the  Marchi  reaction.  It 
was  knowledge  of  this  fact  that  led  us  in  part  to  this  investi- 
gation, and  our  expectation  has  been  fully  confirmed  that 
accompanying  the  Marchi  reaction  in  degenerated  nerve-fibres 
is  a  replacement  of  the  phosphorised  fat  by  non-phosphorised 
fat. 

Before  the  commencement  of  our  joint  work,  Dr  Mott  j-  had 
made  some  preliminary  experiments  in  this  direction,  which 
were  continued  in  conjunction  with  Dr  Barratt.J  Spinal  cords, 
on  one  side  of  which  degeneration  had  occurred,  due  to  a 
lesion  in  the  opposite  cerebral  hemisphere,  were  divided  longi- 
tudinally into  two  halves ;  each  half  was  extracted  with  ether 

*  Zeit.  f.  physiol.  Chem.,  vol.  xxvii.,  p.  370. 

+  Clifford  Allbutt's  System  of  Medicine,  vol.  i.,  Pathology  of  Nutrition. 

\  Proc.  Phys.  Soc.j  Jour,  of  Phys.,  vol.  xxiv.,  p.  iii. 


x.] 


THE  MARC  HI  REACTION 


in  a  Soxhlet's  apparatus.  The  residue  of  the  ethereal  extract 
from  the  degenerated  side  was  more  abundant,  but  contained 
less  phosphorus  than  on  the  healthy  side.  The  degenerated 
half  of  each  cord  was  also  more  watery. 

But  the  Marchi  method  may  be  shortened  and  simplified 
by  placing  the  nerve  into  Marchi  fluid  direct,  without  the 
previous  hardening  for  ten  days  or  so  in  Miiller's  fluid. 

The  result  comparing  the  two  methods  may  be  stated  in 
tabular  form  as  follows: — 


Nerve-Fibres. 

White  Matter  of  Central  Nervous 
System. 

Healthy. 

Degenerated. 

Healthy. 

Degenerated. 

i.  Marchi) 
direct    ./ 

Dark       greyish-\ 
green     .    .     .J 

Black. 

Greyish  -  green,  ^ 
darker       than  1 
with       nerve-  j 
fibres     .     .     J 

Black. 

2.    Marchi,   \ 
after      \ 
Miiller.J 

Greyish  -  green,  \ 
but    not   quite  V 
so  dark.     .     .J 

Black. 

Greyish  -  green,  \ 
like  nerve-fibres  J 

Black. 

The  slight  differences  of  tint  noted  in  the  two  methods  are 
for  practical  purposes  negligible. 

If  now  you  remember  what  I  told  you  on  a  previous  occasion 
about  osmic  acid  and  fat  (p.  67),  you  will  recollect  that  the 
reduction  of  the  osmium  tetroxide  is  due  to  the  presence  of  olein 
or  oleic  acid,  or  some  member  of  the  unsaturated  series.* 

The  unsaturated  acrylic  series  of  fats  represented  by  olein 
is  capable  of  reducing  osmium  tetroxide.  Lecithin  and  other 
phosphorised  fats  of  the  nerve  (see  p.  67)  contain  the  olein 
radical,  and  so  reduce  the  osmium  tetroxide.  But  if  the  osmic 
acid  is  mixed  with  Miiller's  fluid,  a  different  result  is  obtained  ; 
the  potassium  bichromate  of  the  Miiller's  fluid  quickly  diffuses 
into  the  nerve,  and  oxidises  the  olein  radical  of  the  lecithin,  so 
that  when  the  more  slowly  diffusing  osmic  acid  gets  in,  no  call 
is  made  upon  it  to  part  with  its  oxygen  ;  hence  no  reduction  of 

*  See  R.  Wlassak,  Archiv  fiir  Entwickehingsmechanik,  1898,  vol.  vi.7 
p.  453  »  a^s°3  Gustav  Mann,  Physiological  Histology ,  p.  315. 


142  DEGENERATION  AND  REGENERATION  [LECT. 

the  osmic  acid  takes  place,  and  the  nerve  does  not  become 
black. 

In  the  case  of  the  ordinary  neutral  fats,  such  as  are  found 
in  adipose  tissue,  the  amount  of  olein  is  so  great,  or  maybe  it 
is  more  loosely  combined  than  it  is  in  lecithin,  that  preliminary 
or  simultaneous  treatment  with  a  chromic  compound  is  insuffi- 
cient to  satisfy  it,  and  so  osmic  acid  is  reduced,  whether  it  is 
used  alone,  or  in  admixture  with  Miiller's  fluid.  Both  osmic 
acid  and  Marchi's  fluid  therefore  produce  an  intense  black 
staining. 

In  the  case  of  the  degenerated  nerve-fibres,  the  case  is  more 
complex.  Here  the  phosphorised  fat  has  been  broken  up,  and 
the  phosphoric  acid  and  choline  liberated.  Hence  the  olein 
which  remains  is  no  longer  in  firm  combination,  and  so  is  free 
to  behave  as  it  does  in  adipose  tissue.  Added  to  this,  we  have 
the  further  possibility  that  the  olein  of  the  degenerated  medul- 
lary sheath  may  be  more  abundant  than  it  is  in  healthy  lecithin. 
This,  however,  has  not  yet  been  proved. 

We  can  now  pass  on  to  look  at  some  of  the  photographs  of 
our  preparations.  Fig.  22  shows  the  nerve  in  transverse  section 
two  days  after  it  had  been  cut.  It  shows  the  fibrils  of  the  axis- 
cylinder  within  the  medullary  sheath ;  this  nerve  was  excitable, 
and  normal  on  microscopic  examination.  The  photograph  is 
necessarily  printed  in  black  and  white,  but  it  will  be  remem- 
bered that  the  colour  produced  by  the  Marchi  stain  is  a  greyish- 
green.* 

The  next  figure  (Fig.  23)  of  the  nerve  of  a  cat  a  day  later, 
when  the  nerve  had  lost  its  irritability,  shows  a  crinkled  outline 
to  the  medullary  sheaths  which,  however,  still  stain  greyish- 
green.  The  well-defined  fibrillae  of  the  axis-cylinder  are  no 
longer  distinct. 

In  the  third  figure  on  this  plate  (Fig.  24)  we  see  a  transverse 
section  of  the  nerve  eight  days  after  the  operation.  There  is 
not  a  healthy  fibre  left,  and  the  black  staining  with  the  Marchi 
reagent  is  quite  intense.  By  this  time  the  percentage  of  phos- 

*  Several  drawings  representing  the  actual  colours  of  healthy  and 
degenerated  fibres  treated  by  the  Marchi  method  will  be  found  in  the  paper 
by  Mott  and  myself  in  the  Phil.  Trans, 


FIG.  22. 


FIG.  23. 


FIG.  24. 


FlG.  22. — Transverse  section  of  motor  nerve,  53  hours  after  operation.  Method — MARCH l's 
fluid  direct.  In  the  actual  preparation  the  fibrils  of  the  axis  cylinder  are  well  denned. 
700  diameters. 

FlG.  23. — Transverse  section  of  nerve,  3  days  after  operation.     Same  method.     500  diameters. 
FlG.  24. — Transverse  section  of  nerve,  8  days  after  operation.     700  diameters. 


[To  face  page  142. 


FIG.  25. 


FIG.  26. 


FIG.  27. 

FlG.  25. — Longitudinal  section  of  nerve,  99  hours  after  operation.     600  diameters. 
FlG.  26. — Longitudinal  section  of  nerve,  IO  days  after  operation.     600  diameters. 
FlG.  27. — Longitudinal  section  of  nerve,  27  days  after  operation. 

[To  face  page  143. 


x.]  REGENERATION  OF  NERVES  143 

phorus  had  sunk  to  half  the  normal.     The  enlargement  of  the 
lymphatic  channels  alluded  to  on  p.  139  is  also  well  seen. 

The  three  photographs  on  the  next  plate  show  the  appear- 
ances seen  in  longitudinal  view.  Fig.  25  gives  an  early  stage 
in  fragmentation  (four  days  after  operation).  Fig.  26  shows  a 
much  more  pronounced  condition  (10  days  after  operation); 
and  Fig.  27  a  still  later  stage  (27  days  after  the  operation)  by 
which  time  absorption  of  the  fatty  material  has  begun.  The 
large  black  spots  are  fat  cells  from  the  neighbouring  connective 
tissue.  The  exact  correspondence  in  their  black  colour,  with 
that  of  the  droplets  of  degenerated  nerve  fat,  is  naturally  much 
better  seen  in  the  actual  specimen,  but  no  doubt  can  be  realised 
even  from  the  photograph. 

The  next  plate  contains  four  figures,  which  show  the  follow- 
ing points  : — 

Fig.  28  is  a  single  fibre  from  a  teased  preparation  of  a 
nerve,  eight  days  after  it  had  been  cut,  stained  so  as  to  bring 
out  the  division  into  two  of  a  neurilemmal  nucleus. 

Fig.  29  is  a  more  highly  magnified  view  of  Fig.  27  (27-day 
cat).  It  shows  a  chain  of  neurilemmal  cells  filled  with  fatty 
particles.  These  cells,  with  probably  the  assistance  of  phagocytes 
from  the  exterior,  apparently  play  an  important  part  in  devour- 
ing the  degenerated  fatty  material,  and  so  bringing  aboilt  its 
removal. 

In  Figs.  30  and  31  we  have  a  longitudinal  and  transverse 
view  respectively  of  a  regenerated  nerve.  The  small  size  of  the 
new  fibres  will  be  realised  on  comparing  Fig.  31  with  Fig.  22, 
the  magnification  in  the  two  cases  being  the  same. 

Regeneration  of  Nerves 

Our  work  on  degeneration  led  us  more  recently  to  take  up 
the  related  question  of  regeneration. 

From  the  microscopic  study  of  the  distal  portions  of  divided 
nerve-trunks,  we  arrived  at  the  conclusion  that  the  activity  of 
the  neurilemmal  cell  has  some  relation  to  the  development  of 
new  nerve-fibres.  We  have  seen  that  at  an  early  stage  they 
multiply  (Fig.  28),  and  later  appear  to  share  with  phagocytes  in 


1 44  DEGENERA  TION  AND  RE  GENERA  TION          [LECT. 

the  removal  of  the  broken-up  myelin  droplets  (Fig.  29).  Sub- 
sequently they  elongate,  and  look  as  though  they  were  connected 
end  to  end,  thus  leading  to  the  formation  of  what  appear  like 
embryonic  nerve-fibres.  To  suppose  that  they  really  form  new 
axis-cylinders  would  be  against  the  views  of  Waller  and  the  older 
physiologists,  who  taught  that  the  axis-cylinder  is  essentially 
the  branch  of  a  nerve-cell  growing  distalwards  from  the  central 
stump.  Among  recent  writers,  Howell  and  Huber,*  who  have 
used  both  histological  and  experimental  methods  of  observation, 
have  arrived  at  the  conclusion  that  although  the  peripheral 
structures  are  active  in  preparing  the  scaffolding,  the  axis- 
cylinder,  the  essential  portion  of  a  nerve-fibre,  has  an  exclusively 
central  origin.  Our  own  experiments,  which  have  been  made 
on  monkeys  and  cats,  are  at  present  incomplete,  but  the  more 
work  we  have  done  on  the  subject  the  more  have  we  become 
convinced  that  this  view  is  the  correct  one. 

Our  preparations  appear  to  us  to  prove  that  the  manifest 
activity  of  the  neurilemmal  cells  is  related  in  some  degree, 
probably  nutritionally,  to  the  successful  repair  of  a  divided 
nerve.  In  situations  like  the  central  nervous  system,  where  the 
neurilemma  does  not  exist,  not  only  is  the  removal  of  degenerated 
myelin  a  very  slow  process,  but,  as  is  well  known,  regeneration 
does  not  occur. 

The  elongating  and  apparently  continuous  strands  of 
neurilemmal  cells  to  which  we  have  alluded  are  seen  in  Fig.  32 
in  the  next  plate.  It  is,  doubtless,  appearances  of  this  kind 
which  have  led  some  recent  observers  to  the  view  that  new 
nerve-fibres  may  have  a  purely  peripheral  origin.  Ballance  and 
Purves  Stewart  hold  this  view.  They,  however,  relied  exclusively 
on  histological  evidence.  One  method  they  employed  was 
Golgi's,  which  can  hardly  be  considered  for  this  purpose  a  trust- 
worthy one.  It  is  well  known  that  black  streaks  are  produced 
by  this  method  by  structures  which  are  not  nervous  at  all.  A 
strand  that  looks  like  a  nerve-fibre  is  not  really  such,  unless  it 
can  be  experimentally  shown  to  be  excitable  and  capable  of 
conducting  nerve-impulses. 

But  even  careful  histological  examination  will  show  that  the 
*  Jour,  of  Phys.,  1892-93,  vol.  xiii.,  p.  335  j  vol.  xiv,,  p.  i. 


FIG.  29. 


FlG.   28. 


FIG.  31. 


FIG.  30. 


FlG.  28. — Single  fibre  from  nerve  of  cat,  8  days  after  section.      Stained  with  logwood  to  show  divisii 
of  nucleus  of  primitive  sheath.     870  diameters. 

FlG.   29.— High-power  view  of  figure  27,  from    27-day   cat.     This  shows  the  part  played  by  phag 
cytes  and  neurilemmal  cells  in  removing  the  degenerated  fat. 

FlG.  30. — Longitudinal  section  of  regenerated  sensory  nerve,  106  days  after  operation.    700  diametei 
FlG.  31. — Transverse  section  of  the  same.     700  diameters. 


[To 'face  page  144. 


FIG.  32. 


FIG.  33- 

FlG.  32. — Longitudinal  view  of  nerve  from  cat  44  days  after  the  operation;  stained  with  log- 
wood.   The  elongating  neurilemmal  cells  alluded  to  in  the  text  are  seen.    500  diameters. 

FlG.  33. — Transverse  section  of  the  regenerated  nerve  of  cat,  100  days  after  section.     Stained 
by  STRCEBE'S  method.     700  diameters. 


[To  face  page  145. 


x.]  REGENERATION  OF  NERVES  145 

strands  of  neurilemmal  cells  are  situated  outside  the  new  axis- 
cylinder,  and  conceal  it  underneath  or  within  them ;  transverse 
sections  of  the  regenerated  nerve  (as  in  Fig.  33)  reveal  the  axis- 
cylinder  quite  distinct  and  separate  within  the  sheath,  the  nuclei 
of  which  retain  an  abnormal  thickness  for  some  time. 

The  statements  of  clinical  observers,  that  in  man  sensation 
rapidly  returns  after  "  freshening  up  "  and  suturing  together  the 
ends  of  a  nerve  which  has  been  divided  a  long  time  previously, 
would  be  very  valuable  evidence  in  favour  of  the  "  peripheral 
theory"  if  it  were  entirely  trustworthy.  A  case  recently  in 
King's  College  Hospital  was  carefully  observed,  and  throws  a 
useful  light  on  this  subject.  A  short  time  after  the  operation 
was  performed  of  uniting  the  ends  of  the  nerve  together,  the 
man  stated  he  was  again  able  to  feel,  but  these  sensations 
rapidly  subsided,  and  sensation  did  not  really  return  until 
months  later.  The  preliminary  sensation  was  doubtless  sub- 
jective ;  the  "  freshening  up "  of  the  central  end  of  the  nerve 
had  evidently  caused  stimulation  of  the  fibres,  which  lasted  for 
some  hours,  and  the  sensations  so  produced  were  referred  by 
the  patient's  mind  to  the  original  terminals  of  the  fibres.  Such 
patients  are  often  hopeful  of  immediate  cure,  and  this  attitude 
of  mind  may  lead  them  to  assert  that  recovery  has  occurred 
before  it  has  really  done  so. 

Some  of  the  best-attested  cases  of  early  recovery  of  sensa- 
tion after  suture  of  a  previously  divided  nerve  have  been 
recorded  by  R.  Kennedy.*  But  here,  again,  the  genuineness 
of  the  recovery  has  been  doubted ;  in  spite  of  apparent 
sensitiveness  to  such  tests  as  needle-pricks,  there  is  usually, 
in  such  cases,  absolute  anaesthesia  to  the  far  more  delicate 
test  of  stroking  the  hairs  over  the  affected  region. 

The  great  difficulty  of  obtaining  trustworthy  evidence  from 
patients  has  led  Dr  Head  recently  to  divide  and  suture  certain 
nerves  in  his  own  arm  and  observe  the  effects  for  himself.  He 
has  not  yet  published  his  results,  but  from  what  I  have  been 
able  to  gather,  he  has  noticed  no  immediate  recovery  ;  and  the 
time  of  return  of  function  has  coincided  with  those  of  physio- 
logical experiments  on  the  lower  animals.  He  also  showed  the 
*  Phil.  Trans.  Roy.  Soc ,  1897,  vol.  clxxxviii.,  B.,  p.  257. 

K 


146  DEGENERATION  AND  REGENERATION          [LECT. 

difficulty  of  localising  the  stimulus  so  that  it  should  not  affect 
the  hyperaesthetic  marginal  zone  of  the  anaesthetic  region ;  this 
probably  explains  the  apparent  early  recovery  to  the  needle- 
prick  test  just  alluded  to. 

Bethe,  who  is  another  exponent  of  the  peripheral  theory, 
asserts  that  the  peripheral  ends  of  cut  nerves  may  be  excitable 
without  union  with  the  central  end ;  but  he  does  not  seem  to 
have  excluded  a  fallacy  which  was  pointed  out  by  Langley  and 
Anderson  at  a  recent  meeting  of  the  Physiological  Society.* 
These  observers  showed  that  in  spite  of  the  absence  of  any 
obvious  connecting  strand  with  the  central  end,  new  nerve-fibres 
had  found  their  way  by  devious  channels  into  the  peripheral 
stump  from  nerves  in  skin  and  muscle  cut  through  in  the 
operation. 

We  have  sought  to  obviate  such  sources  of  fallacy  by  the 
following  means : — 

The  incisions  were  made  as  small  as  possible,  and  the  parts 
separated  from  the  nerve-trunks  with  as  little  cutting  as,  possible. 
In  cats,  one  incision  over  the  buttock  allowed  us  to  divide  the 
sciatic  nerve  high  up.  Another  in  the  ham  enabled  us  to  divide 
the  two  popliteal  nerves.  The  intervening  portion  of  the  sciatic 
nerve,  about  4  or  5  inches  long,  can  be  easily  pulled  out. 
Additional  security  to  prevent  union  with  central  fibres  was  in 
some  cases  obtained  by  enclosing  the  upper  end  of  each 
popliteal  nerve  in  closed  caps  made  out  of  small  drainage  tubes 
about  half  an  inch  long.  A  period  of  one  hundred  to  one 
hundred  and  fifty  days  was  then  allowed  to  elapse,  in  order  that 
if  regeneration  was  going  to  occur  in  the  peripheral  segments  of 
the  nerve,  it  might  have  an  opportunity  of  doing  so.  At  the 
end  of  this  time  the  animal  was  anaesthetised,  and  the  nerves 
tested  by  electrical  stimulation.  In  all  cases  they  were  entirely 
inexcitable  to  strong  faradic  currents,  and  the  wasted  muscles 
also  had  largely  lost  their  power  of  response  to  this  form  of 
stimulation.  To  the  naked  eye  the  nerves  were  pale.  The 
animals  were  killed,  and  microscopical  investigation  of  the 
nerves  showed  no  trace  of  regeneration.  In  those  cases  where 

*  Proc.  Phys.  Soc.,  I3th  December  1902.  Jour,  of  Phys.,  vol.  xxix., 
p.  ii.  See  more  fully,  ibid.,  1904,  vol.  xxxi.,  p.  418. 


x.]  REGENERATION  OF  NERVES  147 

the  nerves  had  been  placed  in  tubes,  it  was  very  difficult  to 
recognise  any  nervous  structure  whatever. 

Another  experiment  suggested  to  us  by  Professor  Gotch 
has  been  performed  both  on  the  monkey  and  cat.  A  large 
nerve  was  divided,  and  the  ends  sutured  together.  After  a 
sufficient  length  of  time  had  elapsed,  restoration  of  function  led 
us  to  suppose  that  regeneration  had  occurred.  The  nerve  was 
exposed  ;  the  union  of  the  two  ends  was  found  to  have  been 
accomplished,  and  the  nerve  was  excitable  both  above  and  below 
the  junction.  A  piece  of  nerve  was  then  excised  an  inch  or  so 
below  the  junction,  and  on  histological  examination  of  this,  all 
traces  of  degenerated  products  were  found  to  have  disappeared, 
and  it  was  made  up  of  fine  new  nerve-fibres,  many  of  which  had 
acquired  a  delicate  medullary  sheath.  After  this  second  opera- 
tion, the  wound  was  closed  and  the  animal  allowed  to  live  for 
ten  days  longer.  It  was  then  killed,  and  the  nerve  both  below 
and  above  the  second  cut  was  then  examined.  No  degenera- 
tion was 'found  in  the  nerve-fibres  above  the  second  lesion,  but 
Wallerian  degeneration  was  shown  by  the  Marchi  method  to 
have  occurred  in  medullated  fibres  of  the  peripheral  portion, 
which  was  quite  inexcitable.  The  direction  of  degeneration  is 
the  direction  of  growth ;  so  this  experiment  shows  that  the 
growth  of  the  new  fibres  had  not  started  from  the  periphery 
centralwards,  but  in  the  reverse  direction. 

Another  piece  of  evidence  bearing  in  the  same  direction 
consisted  in  examining  regenerated  nerve-fibres  in  various  parts 
of  their  course.  We  think  in  some  cases  that  the  more  distant 
the  situation  from  the  original  point  of  section,  the  less  perfectly 
developed  the  new  fibres  appear  to  be ;  myelination  has  pro- 
gressed less  in  the  distal  portion  of  their  course.  But  further 
observations  on  this  point  are  being  made. 

Another  set  of  experiments  consisted  in  attempting  to 
ascertain  the  influence  of  stimulus  on  regeneration.  A  monkey's 
arm  was  rendered  immobile  by  the  division  of  a  number  of  the 
upper  posterior  roots.  The  anterior  cornual  cells,  from  which 
the  corresponding  motor  fibres  originate,  are  thus  not  subjected 
to  stimuli  from  the  periphery;  and,  as  Mott  and  Sherrington 
were  the  first  to  show,  the  arm  is  as  much  paralysed  as  if  the 


148  DEGENERATION  AND  REGENERATION          [LECT. 

anterior  roots  had  been  cut.  We  have,  however,  again  noticed 
in  some  of  these  animals  under  the  influence  of  strong  emotion 
(for  instance,  when  the  monkey  is  prevented  from  reaching  with 
the  sound  hand  a  piece  of  apple),  that  some  efforts  are  made  to 
move  the  other  limb.  When  the  animal  is  living  in  its  cage 
under  ordinary  conditions,  it  makes  no  effort  to  move  the  limb, 
which  in  successful  experiments  (i.e.  when  a  sufficient  number  of 
roots  have  been  entirely  cut  through)  hangs  helpless  like  a  flail. 
A  large  nerve  in  the  arm  (median  or  ulnar)  was  then  divided, 
and  the  ends  sutured  together ;  the  corresponding  ,nerve  was 
divided  and  sutured  on  the  non-paralysed  side  as  a  control 
experiment.  The  animal  was  finally  killed  ;  the  interval  between 
the  operation  and  death  varied  in  different  experiments,  but  the 
best  time  for  making  the  observation  we  finally  determined  to 
be  between  sixty  and  seventy  days  after  the  nerves  had  been 
cut. 

Union  of  the  divided  nerves  occurs  on  both  sides  of  the 
body,  and  in  our  early  experiments,  the  nerve  on  the  side 
corresponding  to  that  on  which  the  posterior  nerve-roots  had 
been  divided  was  found  to  be  less  excitable  to  the  faradic 
current  ;  histologically,  this  nerve  showed  a  looser  texture,  and 
new  nerve-fibres,  though  present,  were  somewhat  less  numerous 
than  on  the  control  side.  In  these  early  experiments,  also,  we 
found  that  the  posterior  cornual  cells  in  the  cervical  region  were 
atrophied,  and  that  there  was  a  considerable  overgrowth  of 
neuroglia  tissue  in  the  posterior  horn. 

Further  examination  (by  the  methylene-blue  and  erythrosin 
stain)  of  these  spinal  cords  showed,  however,  that  there  had 
been  a  considerable  number  of  small  haemorrhages,  sufficient 
in  some  cases  to  cause  degeneration  in  various  descending 
tracts  in  the  cord.  It  therefore  became  quite  possible  to 
explain  the  effects  observed  by  this  complication.  We  are 
inclined  to  think  that  the  haemorrhages  are  not  due  to 
mechanical  injury  of  the  cord  during  the  operation,  but  are 
to  be  explained  by  the  loss  of  support  in  the  cord  tissue 
which  follows  degeneration  of  the  entering  posterior  root- 
fibres. 

In  several    of    the   later   experiments,    in   which   the   cord 


x.]  REGENERATION  OF  NERVES  149 

haemorrhages  did  not  occur  to  any  great  extent,  we  have  been 
unable  to  detect  any  marked  changes  in  the  posterior  cornual 
cells,  or  any  difference  to  stimulation  or  in  microscopical 
structure  between  the  regenerated  nerves  of  the  two  sides. 

This  result  accords  with  some  experiments  carried  out  by  Anderson  *  on 
developing  animals ;  he  found  that  section  of  all  the  posterior  roots 
connected  with  a  limb"  caused  no  retardation  in  the  development  of  the 
corresponding  anterior  roots. 

In  further  experiments  we  sought  to  cut  off  the  cerebral 
influence  by  removing  the  cortical  arm  area  of  the  opposite  side, 
in  addition  to  dividing  posterior  roots  as  before.  In  this  case, 
also,  the  regenerated  nerves  of  the  two  sides  were  equally 
responsive  to  stimulation^  and  histological  evidence  of  any  marked 
difference  between  them  was  also  lacking. 

At  present  we  are  engaged  in  performing  experiments  in 
which  a  transverse  section  of  the  cord  is  combined  with  division 
of  posterior  nerve-roots.  By  this  means  we  hope  to  still  further 
reduce  the  action  of  innervation  currents  on  the  anterior  cornual 
neurons  by  cutting  off  the  stimuli  which  enter  by  the  posterior 
roots,  as  well  as  those  which  descend  from  the  brain.  It  is 
quite  possible  that  the  paths  which  will  even  under  those 
circumstances  remain  open  (commissural  and  association  tracts) 
may  be  sufficient  to  maintain  the  activity  of  the  anterior  cornual 
cells  in  the  sprouting  forth  of  new  axons  in  a  peripheral  nerve, 
though  they  may  be  insufficient  to  induce  those  cells  to  send 
effective  impulses  along  them. 

Warrington  has  stated  that  when  posterior  nerve-roots  are 
cut,  the  anterior  nerve-cells  undergo  the  chromatolytic  change 
associated  with  inactivity.  Warrington's  observations,  how- 
ever, were  made  at  an  early  date  after  the  division  of  the  roots. 
In  the  animals  which  we  have  killed  at  the  late  dates  mentioned, 
it  was  not  possible  with  any  certainty  to  tell  by  looking  at  the 
anterior  horn  cells  of  the  two  sides  which  was  the  side  on  which 
the  posterior  roots  had  been  divided. 

Another  question,  and  one  which  is  of  equal  interest  in  any 
I  have  mentioned,  is  the  mode  of  origin  of  the  medullary  sheath. 

*  Jour,  of  Phys.)  vol.  xxviii.,  p.  499. 


150  DEGENERATION  AND  REGENERATION  [LECT. 

This,  however,  I  must  only  touch  upon.  The  activity  of  the 
neurilemmal  cells  is  the  only  point  in  favour  of  the  view  that  it 
is  formed  from  them.  All  the  other  facts  point  to  the  origin 
of  the  medullary  sheath  from  the  axis-cylinder,  and  this  evidence, 
which  to  me  seems  conclusive,  is  : — 

1.  In    a   developing   or   regenerating  nerve-fibre,  complete 
functional   activity  is   associated  with   the   appearance   of  the 
medullary  sheath. 

2.  When  a  nerve  is  cut,  the  medullary  sheath  is  a  part  which 
markedly  shares  in  the  degenerative  process. 

3.  The  medullary  sheath  appears  in  the  nerve-fibres  of  the 
central    nervous  system,  that  is,  in  a  portion  of  their  course 
where  the  primitive  sheath  is  absent. 

The  axis-cylinder  and  its  sheaths  must  necessarily,  for  de- 
scriptive purposes,  be  considered  separately.  There  is  little  doubt 
in  my  own  mind,  that,  functionally,  all  three  parts  of  a  nerve- 
fibre  must  be  considered  to  act  as  an  organic  whole,  with 
intimate  inter-relations  of  a  nutritional  or  metabolic  nature. 

The  statements  by  Bethe,  Dohrn  and  others  concerning  the  peripheral 
origin  of  new  nerve-fibres  has  called  forth  a  vigorous  protest  from  the  veteran 
histologist,  Kolliker,  in  a  recent  number  of  the  Anat.  Anzeiger  (vol.  xxv., 
p.  i).  He  points  out  that  Ramon  y  Cajal's  observations  support  the  view  of 
the  central  origin  of  nerve-fibres,  which  he,  among  others,  enunciated  many 
years  ago.  The  process  of  the  nerve -cell  which  we  call  the  axon  becomes 
differentiated  into  axis-cylinder  and  medullary  sheath.  The  investing  cells 
that  form  the  neurilemma  are  mesoblastic  in  origin. 

It  would  on  d  priori  grounds  be  extremely  improbable  that  mesoblastic 
cells  should  be  capable  of  forming  new  axis-cylinders,  and  assuming  the 
highly-specialised  function  of  conducting  nerve  impulses  in  place  of  the 
original  axis-cylinders,  which  have  an  epiblastic  origin. 

This  brings  me  to  the  end  of  what  I  have  to  say.  The 
researches  are  many  of  them  unfinished,  but,  nevertheless,  my 
lectures  must  terminate;  and  I  have  in  conclusion  to  express  my 
gratitude  to  those  who  have  given  me  the  opportunity  of 
stringing  my  facts  together  in  this  way,  and  to  you  who  have 
patiently  and  attentively  followed  my  utterances. 

In  the  conventional  works  of  fiction,  the  author  is  able  to 
manipulate  his  puppets  in  such  a  way  that  the  wind-up  is 
satisfactory,  and  all  live  happily  ever  afterwards.  In  actual  life, 


x.]  REGENERA  TION  OF  NER  VES  \  5 1 

things  seldom  happen  so  as  to  terminate  in  a  round-up  of  this 
kind.  So  it  is  with  science ;  its  great  charm  is  that  it  never 
finishes.  We  call  a  halt  now  and  then  and  report  progress, 
but  work  goes  on,  new  discoveries  are  made,  and  every  real 
discovery  opens  the  road  to  others,  and  still  others  beyond 
those. 


INDEX 


ABSENCE  of  fatigue-changes  in  nerve- 
fibres,  89 

Acid,  carnic,  32,  46,  56  ;  glycero-phos- 
phoric,  66 ;  inosinic,  32,  41 ;  kephalic, 
69  ;  lactic,  32,  37  ;  methyl  amino- 
acetic,  44  ;  oleic,  66,  67,  141 ;  osmic, 
67,  141  ;  oxypropionic,  37  ;  palmitic, 
66  ;  phospho-molybdic,  phospho- 
tungstic,  123  ;  protic,  40 ;  sarco- 
lactic,  39,  51  ;  stearic,  66;  uric,  32, 

44 

Activity  of  nerve,  hypothetical  pro- 
duction of  carbon  dioxide  during, 

82 

Adenine,  63 
Agaricus  muscarius,  67 
Albumin,  21 
Alcock,     galvanometer     experiments, 

95,  103-105 

Alcohol  as  a  proteid  precipitant,  21 
Alcoholic  neuritis,  nerve  degeneration 

in,  129 

Alcoholic  psychosis,  132 
Allen  and  French,  on  choline  in  blood, 

129 

Amido-myelin,  68 
Amphi-creatine,  43 
Amylolytic  ferment  in  muscle,  31 
Anasmia,  cerebral,  79,  89,  101 
Anderson,  on  nerve  regeneration  and 

growth,  146,  149 
Animal  gum,  53 
Ansiaux,  on  heat  coagulation  of  pro- 

teids,  108 

153 


Antipeptone,  46 

Araki,  on  sarcolactic  acid,  40 

Athletes,  and  training,  34 

Atrophy,   muscular,  133 ;    disuse,   88, 

134 
Axis-cylinder,  growth  of,  144,  145,  149 

BALKE,  on  carnic  acid,  46 
Ballance,  on  nerve  regeneration,  144 
Barratt,  on  nerve  degeneration,  140 
Bastian,  on  specific  gravity  of  brain,  59 
Battistini,    on     reaction    of    nervous 

tissues,  8 1 
Baumstark,    on   proteids   of   nervous 

tissues,  62 

Bellows  recorder,  92,  94 
Beri-beri,  131 
Betaine,  67 

Bethe,  on  nerve  regeneration,  145 
Birds,  muscles  of,  20 ;  nerves  of,  107, 

H5 

Blankenhorn,  on  protagon,  64 

Blood,  choline  in  the,  127,  135 

Blood-clotting,  and  muscle-clotting, 
their  analogy,  5 

Blood-proteids,  23 

Bohm,  on  sarcolactic  acid,  39 

Boileau,  on  specific  gravity  of  brain,  59 

Brain,  specific  gravity  of,  58 

Brain  tissue,  influence  of  age  on  per- 
centage of  water  in,  58 

Brains  of  cats,  influence  of  high 
temperature  on,  111-113 

Brieger,  on  neurine,  67 


154 


INDEX 


Brodie,  T.  G.,  on  heat  rigor,  14-16, 
19,  54 ;  on  perfusion  of  isolated 
kidney,  21  ;  experiments  on  splenic 
nerves,  82,  91  ;  his  bellows  re- 
corder, 92 

Browne,  Sir  James  Crichton  (Sex  in 
Education),  on  specific  gravity  of 
brain,  58 

Briicke,  on  muscle  carbohydrates,  33 

Brunton  and   Rhodes,  on  glycolysis, 

31 
Bunge's  analysis  of  muscle  salts,  47 

CALCIUM,  51 

Calcium  salts,  essential  in  blood- 
clotting,  5 

Capillary  pressure  in  brain,  72 

Carbohydrates,  colloidal  and  crystal- 
line, 21  ;  of  muscle,  33 

Carbon  dioxide,  51;  hypothetical 
production  of,  during  the  activity  of 
nerve,  82 

Cardiac  muscle,  3 

Cardio-inhibitory  fibres,  90 

Carnic  acid,  32,  46,  56 

Carniferrin,  46 

Carnine,  32,  41 

Carnosine,  41 

Caseinogen,  24 

Cats,  experiments  on  brains  of,  m- 
113  ;  sciatic  nerves  of,  137 

Cavazzani,  on  cerebro-spinal  fluid,  73 

Cell-globulin,  coagulation  of,  107 

Cell-protoplasm,  101 

Cells,  nerve-,  79  ;  anterior  horn,  149  ; 
methods  of  examining,  85,  87-89 

Centres,  nerve-,  79,  85 

Cerebral  anaemia,  79,  89,  101 

Cerebrins  or  cerebrosides,  60,  64, 
69 

Cerebron,  70 

Cerebro-spinal  fluid,  70,  117,  118; 
osmotic  relationships  of,  77 ;  in 
degenerative  diseases  of  nervous 
system,  127 


Chemical,  changes  accompanying  the 
contraction  of  muscle,  49  ;  composi- 
tion of  nervous  tissues,  57  ;  exami- 
nation of  nerves,  136  ;  pathology  of 
General  Paralysis  of  the  Insane,  1 16; 
reactions  of  choline  and  neurine,  123  ; 
tonus,  49 

Chemistry  of  tendon,  52 

Cholesterin,  60,  64,  65 

Choline,  64,  66,  67,  76,  83-85,  1 17,  1 18  ; 
chemical  reactions  of,  123-127  ;  in 
the  blood,  127,  135,  138 

Chondro-mucoid,  54 

Chromatolysis,  87,  88 

Chromoplasm,  87,  88 

Cleghorn,  on  sympathetic  ganglia,  85 

Coagulation  temperature,  16,  102-115 

Cold,  effect  of  on  nerve,  103 

Cold-blooded  and  warm-blooded  ani- 
mals compared  in  relation  to  muscle, 
14,  20  ;  to  nerve,  106,  107,  no,  114 

Combined  sclerosis,  129 

Comparative  chemistry  of  liver,  107.; 
of  muscle,  1 1,  14,  20  ;  of  nerve,  106, 
no,  114 

Connective  tissues,  52 

Contractile  tissues,  48 

Corin,  on  heat  rigor,  108 

Cramer,  on  pseudo-cerebrin,  70;  on 
choline,  85 

Creatine,  creatinine,  32,  42-44 

Croftan,  on  bile  salts  in  blood,  128 

Cruso-creatinine,  43 

DANA,  examination  of  cerebro-spinal 

fluid,  132 

Degeneration  of  nerves,  99,  134,  139 
Degenerative     diseases     of    nervous 

system,  cerebro-spinal  fluid  in,  127 
Delezenne,  on  blood-clotting,  30 
Demoor,  on  dendrites,  97 
Dendrites,  97,  99 
Dextrin,  21,  32 

Dextro-rotatory  lactic  acid,  38 
Dextrose,  36,  51 


INDEX 


155 


Diaconow,  on  protagon,  64 

Dioxypurine,  45 

Diphasic  variation,  103 

Disseminated  sclerosis,  129,  133 

Disuse  atrophy,  88,  134 

Dixon,  on  spermine,  128 

Dogfish,  urea  in  blood  of,  42 

Donath,  on  cerebro-spinal  fluid,  125, 
128,  132,  133 

Dormeyer,  on  muscle  fat,  36 

Du  Bois  Reymond's  negative  varia- 
tion, 102 

Dynamograph,  Waller's,  86 

EDMUND'S  intestinal  oncometer,  121 
Egg-white  proteids,  24 
Ehrlich,  methylene  blue,  79 
Elastin,  54 

Electrometer,  capillary,  95 
Endogenous  uric  acid,  45 
Enzymes,  see  Ferments 
Ergographs,  86 
Ethylene  lactic  acid,  37 
Euglobulin,  20-25 
Eve,  on  fatigue,  88,  90,  104 
Exogenous  uric  acid,  45 
Extractives,  and  salts  of  muscle,  32  ; 
of  nerve,  60 

FARADIC  excitation  of  splenic  nerve, 

92-94 
Fatigue,  of  muscle,  85,  86  ;  central  and 

peripheral,  86-89  >  stimulation,  95 
Fatigue     changes     in     nerve  -  fibres, 

absence  of,  89 
Fat,  of  muscle,  32,  36 
Fats,  phosphorised,  60,  63 
Ferments  of  muscle,  30 
Fibres,  vaso-constrictor,  cardio-inhibi- 

tory,  and  non-medullated,  90 
Fibres,  nerve-,  79,  86  ;  and  Wallerian 

degeneration,  139 
Fibrin  ferment,  30 
Fibrinogen,  5,  22 
Fletcher,  on  rigor  mortis,  \  2 


Folin,  O.,  on  the  coagulation  theory  of 

rigor  mortis,  5 
French  and  Allen,  on  choline  in  blood, 

129 

Frerichs,  on  urea,  42 
Freytag,  on  protagon,  65 
Functional  neuroses,  129,  133 
Fiirth,  von,  on  muscle  plasma,  6,  8, 

10  ;  on  lactic  acid,  39 

GALVANOMETRIC  response  of  nerve  to 

stimulation,  influence  of  temperature 

on,  102 
Gamgee,    on    protagon,    64,   70 ;    on 

pseudo-cerebrin,  69 
Ganglia,  sympathetic,  85 
Gautier,  on  creatinines,  43 
Gelatin,  53,  54,  60 

General  Paralysis  of  the  Insane,  116 
Gies,     William     J.,     on     connective 

tissues,  53 

Gleiss,  on  muscle,  39 
Globulin,    21,    62,    63 ;     coagulation 

temperature  of  cell-,  107 
Glycerin,  66 

Glycero-phosphoric  acid,  66 
Glycine,  40 
Glycogen,  21,  32-35 
Glycolytic    ferments    and    glycolysis, 

3i 

Gluco-proteids,  53 
Glucosamine,  53 
Golgi's  silver-chrome  method,  87,  97, 

99,  144 
Gompertz,  on  specific  gravity  of  brain, 

59 

Gotch,  experiments  with  capillary 
electrometer,  95  ;  on  nerve  regen- 
eration, 147 

Gottwalt,  on  proteids,  21 

Gravity,  specific  of  brain,  58 

Griinbaum,  Otto,  chemical  test  for 
choline  in  blood,  128,  132 

Gscheidlen,  on  reaction  of  axis- 
cylinder,  8 1 


156 


INDEX 


Guanine,  63 
Gum,  animal,  53 

Gumprecht,    on    cerebro-spinal   fluid, 
77,  83,  132 

HAEMOGLOBIN,  9,  28,  29 
Haemochromogen,  28,  29 
Haemorrhage  in  the  spinal  cord,  148 
Haiser,  on  inosinic  acid,  41 
Hammarsten,  on  sarcolactic  acid,  40 
Hastings,  on  choline  in  cerebro-spinal 

fluid,  132 

Head,  on  nerve  regeneration,  145 
Heat   contraction   in   nerve,   105  ;    in 

liver,  107 

Heat  rigor  of  muscle,  13-25 
Hedin,  on  proteolytic  ferments,  12 
Heidenhain's  lymph  theory,  74,  8r 
Helmholtz,   on   chemical   changes   in 

muscle,  50 

Hepato-globulin,  62,  63,  108 
Hermann's  inogen  theory,  50,  51 
Herpes  zoster,  132 

Hewlett,  on  heat  coagulation,  17, 19, 1 08 
Hill,  Leonard,  on  cerebro-spinal  fluid, 

71  ;  on  oxygen  in  brain,  79 
Histo-haematins,  27 
Histological  examination  of  nerve-cells, 
evidence   of   metabolic  activity    of 
nervous  centres  derived  from,  85,  139 
Hoppe-Seyler,  on  sarcolactic  acid,  39  ; 

on  creatinine,  43  ;  on  protagon,  64 
Howell,  on  stimulation  fatigue,  95  ;  on 
nerve  temperature,   104 ;   on  nerve 
regeneration,  144 
Huber,  on  nerve  regeneration,  144 
Hydrocephalus,  75,  76 
Hyperpyrexia,  107 
Hyperthermia,  no 
Hypothetical    production    of    carbon 

dioxide  during  activity  of  nerve,  82 
Hypoxanthine,  32,  44 
Hysteria,  132 

IMOGEN,  50,  51 


Inorganic    salts    of   muscle,    47  ;     of 

nerve,  60 

Inosinic  acid,  32,  41 
Inosite,  32 
Insane,  chemical  pathology  of  General 

Paralysis  of  the,  116 
Involuntary  muscle,  9 
Iodine  test  for  choline,  123,  129 
Ions,  48 
Irritability     of    muscle,    temperature 

necessary  to  destroy,   19  ;  of  nerve, 

permanent  loss  of,  104 
Iso-creatinine,  44 
Isomerides,  37,  38 

JOACHIM,    J.,    on   serum  globulin   in 

urine,  25 
Johnson,  G.  S.,  method  of  estimating 

creatinine,  43 

KATZ,  J.,  on  muscle  salts,  47 

Kemmerich,  on  creatinine,  43 

Kennedy,  on  nerve  regeneration,  145 

Kephalic  acid,  69 

Kephalin,  64,  65,  68 

Kerasin,  69 

Kinetoplasm,  87 

Koch,  Waldemar,  on  kephalin,  64  ; 
on  phosphorised  fats,  68 

Kolliker,  on  nerve  regeneration,  150 

Kossel,  on  protagon,  65 

Kriiger,  on  carnic  acid,  46 

Krukenberg,  on  urea  in  muscle,  42 

Kiihne,  muscle  plasma,  4  ;  on  sarco- 
lactic acid,  39  ;  his  antipeptorie,  46 

Kiilz,  on  muscle  carbohydrates,  33 

Kutscher,  on  antipeptone,  46 

Kyes,  Preston,  on  lecithin,  133 

LACT-ALBUMIN,  24 

Lactic  acid,  32,  37  ;  optically  inactive, 

37  ;    dextro-rotatory,    laevo-rotatory, 

38 

Laevo-rotatory  lactic  acid,  38 
LangendorfF,  O.,  on  reaction  of  nervous 

tissues,  8 1 


INDEX 


Langley,  on  nerve  regeneration,  145 
Latham,  on  proteid  molecules,  40 
Leathes,  on  muscle  fat,  37 
Lecithans,  64 

Lecithin,  40,  60,  63-68,  133,  135 
Lesem  and  Gies,  on  protagon,  65 
Levene,  P.  A.,  on  proteids  of  nervous 

tissues,  63 

Levy,  on  myohaematin,  29 
Lewandowsky,   M.,  on  cerebro-spinal 

fluid,  77 
Liebig,   on  muscle   proteids,   34 ;   on 

inosinic  acid,  41 
Liebreich,  on  protagon,  64 
Lipochromes,  27 
Liver,  removal  of,  35 
Liver,  heat  contraction  of,  107 
Lugaro,  on  sleep  and  narcosis,  97 
Lutein,  27 

MACALLUM,  A.  B.,  test  for  potassium 

in  tissues,  48,  60 

MacMunn,  on  muscle  pigments,  27-29 
Mall,  on  retiform  tissue,  54 
Maltose,  36 
Marchi's  method  of  staining,  116,  134, 

138,  140-142,  147 
Marinesco,  on    kinetoplasm,  87,  no, 

114 

Medullary  sheath,  origin  of,  149 
Metabolic   activity   in   nervous  struc- 

tures^  83  ;  in  nervous  centres,  85 
Metabolism,  in  nervous  tissues,  78-85 
Methyl  amino-acetic  acid,  44 
Methylene  blue,  79,  87,  99 
Milk  proteids,  24 

Mineral    constituents    and    muscular 
f      contraction,  48 
Minkowski,  on  removal  of  liver,  35 
Molecules,  proteid,  17,  1 8 
Moleschott,  S.,  on  reaction  of  nervous 

tissues,  8 1 

Moore,  B.,  on  anaesthesia,  100 
Morner,  on  muscle  pigments,  29  ;  on 

creatinine,  43 


Mosso's  ergograph,  86 

Mott,  on  pathological  questions,  76, 
108,  1 10,  131,  140,  147 

Mucin,  52,  53 

Mucoid,  53 

M  tiller,  formula  for  cerebrin,  69  ;  on 
reaction  of  nervous  tissues,  82  ;  fluid, 
140-142 

Muscarine,  67 

Muscle,  carbohydrates  of,  33  ;  changes, 
80  ;  dead  and  living,  51  ;  extrac- 
tives and  salts  of,  32  ;  fat  of,  36  ; 
fatigue,  86,  87;  ferments  of,  31; 
general  composition  of,  3  ;  proteids 
of,  3-11  ;  serum  and  plasma,  4,  5  ; 
irritability  of,  19  ;  inorganic  salts  of, 
47  ;  pale  and  red,  26  ;  paralysis  of, 
35  ;  pigments  of,  26  ;  proteids,  24  ; 
sugar  in,  35  ;  urea  in,  41 

Muscle-clotting  and  blood  -  clotting, 
their  analogy,  5 

Muscular,  atrophy,  1 33 ;  contraction  and 
mineral  constituents,  48  ;  chemical 
changes  accompanying  contraction, 
49  ;  classification  of  fibres,  3 

Myelin,  68 

Myo-albumose,  9 

Myogen-fibrin,  10 

Myohasmatin,  27-29 

Myoproteid,  u 

Myosin,  4,  5,  8,  10 

Myosinogen,  5,  8,  n,  14,  24 

Myxoedema,  52 

NARCOSIS,  96 

Nasse,  O.,  sugar  in  muscle,  36  ;  origin 
of  sarcolactic  acid,  39 

Nawratski,  on  cerebro-spinal  fluid,  75 

Nawrocki,  on  creatinine,  43 

Negative  variation,  102 

Nerve,  galvanometric  response  of,  to 
stimulation,  102  ;  hypothetical  pro- 
duction of  carbon  dioxide  during 
the  activity  of,  82  ;  heat  contraction 
in,  105  ;  loss  of  irritability  of,  104 


158 


INDEX 


Nerve-cells,  79,  85  ;  methods  of  ex- 
amining, 85,  87-89  ;  anterior  horn, 
149 

Nerve-centres,  79,  85 

Nerve-fibres,  79,  86 ;  absence  of 
fatigue  change  in,  89 ;  Wallerian 
degeneration  of,  139  ;  regeneration, 

H3 

Nerve-proteids,  61,  102 

Nerve-roots,  posterior,  149 

Nerves,  chemical  examination  of,  136  ; 
histological  examination  of,  139  ; 
degeneration  and  regeneration  of, 
134  ;  medullated,  93  ;  non-medul- 
lated,  92,  93,  96  ;  splenic,  91,  94,  95  ; 
union  of  divided,  148  ;  vaso-motor, 

94 
Nervous  system,  percentage  of  water 

in,   58  ;   cerebro-spinal  fluid  in  de- 
generative diseases  of,  127 
Nervous  tissues,  chemical  composition 

of,    57  ;    solids  of,  60 ;   proteids  of, 

61-63,  I02  5    metabolism  in,   78-85  ; 

reaction  of,  81 
Neubauer,  on  creatinine,  43 
Neurilemma,  139 
Neurilemmal  cells,  144 
Neurine,  67  ;  physiological  action  of, 

118  ;  chemical  reactions  of,  123 
Neuritis,  nerve  degeneration  in,  129, 

130 

Neuroglia,  139 
N euro-globulin,  62,  108 
Neuro-keratin,  60,  62 
Neurons,  97 

Neuroses,  functional,  129,  133 
Nissl,    methylene  -  blue  process,   87  ; 

granules,  88,  89,  99 
Nitrogenous  extractives  of  muscle,  32, 

40 
Noll,  study  of  central  ends  of  divided 

nerves,  140 

Non-medullated  nerves,  96 
Non-nitrogenous  extractives  of  muscle, 

32 


Nuclein,  45,  60 

Nucleo-proteids,   9,    29,    62,    63,    117, 

126 
Nucleon,  46 

OBERSTEINER,  on  sleep,  73,  96 

Olein,  oleic  acid,  66,  141 

Oncometer,  92,  120 

Optically  inactive  lactic  acid,  37 

Ord,  on  myxcedema,  52 

Organic  solids  in  cerebro-spinal  fluid, 

73 
Osborne,  on  lactic  acid,  39  ;  on  nervous 

tissues,  85 
Osmic  acid   (osmium    tetroxide),   67, 

141 

Osseo-mucoid,  54 
Oxidases,  50 
Oxygen  in  muscular  metabolism,  50  ; 

in  nervous  metabolism,  79 
Oxyhaemoglobin  in  muscle,  26 
Oxypropionic  acid,  37 
Oxypurine,  45 

PALMITIC  acid,  palmitin,  66 

Panormoff,  on  sugar  in  muscle,  35 

Paralysis,  of  muscle,  35  ;  of  the 
Insane,  chemical  pathology  of,  116 

Paramyelin,  68 

Para-myosinogen,  8,  n,  14,  24,  108 

Pauli  and  Rona,  on  precipitation  of 
colloids,  22 

Pavy,  on  muscle  carbohydrates,  33 

Penicillium  glaucum,  38 

Pepsin,  12 

Peripheral  theory  of  nerve  regenera- 
tion, 145 

Petrowsky,  on  proteids  in  nervous 
tissues,  62 

Pfliiger,  on  muscle  carbohydrates,  33 

Phagocytes,  116,  143 

Phosphatides,  64 

Phospho-molybdic  acid,  123 

Phosphorised  fats,  60-63 

Phosphorus  in  nerve,  136,  138,  139 


INDEX 


159 


Phospho-tungstic  acid,  123 

Phrenosin,  69 

Physiological  action   of    choline   and 

neurine,  118 
Pigments  of  muscle,  26 
Plasma,  muscle,  4,  5 
Platino-chlorides,  123-125,  128 
Platinum  test  for  choline,  124,  135 
Pressure,  capillary,  in  brain,  72 
Protagon,  60,  63-65 
Proteid  molecules,  17,  1 8 
Proteids,  53  ;  nerve-,  102  ;  of  muscle, 

3-11,    24;   typical  groups  of  blood, 

egg-white,  milk,  22-24  ;   of  nervous 

tissues,  60-63 
Proteolytic  enzymes,  31 
Protic  acid,  40 
Przibram,  Hans,  on  muscle  proteids, 

ii 

Pseudo-cerebrin,  69,  70 
Pseudo-globulins,  20-25 
Pseudo-xanthine,  43 
Psychosis,  alcoholic,  132 
Purine  substances,  45 
Pyrocatechin,  75 

RANSOM,  F.,  on  cerebro-spinal  fluid, 

77 

Reaction  of  nervous  tissues,  81 
Regeneration  of  nerves,  134,  143 
Relation  of  water  and  solids  in  muscle, 

3  ;  in  nervous  tissue,  57 
Reticulin,  54-56 
Retiform  tissue,  54 
Richardson,  on  heat  rigor,  14,  109 
Rigor  mortis,  4,  5,  1 1 
Rigor  of  muscle,  heat,  13-25 
Roaf,  H.  E.,  01  anaesthesia,  100 
Rohmann,  on  lactic  acid,  39 
Rolleston,  on  nerve  temperature,  80 

SALTS   of  muscle,    inorganic,  47  ;    of 

nerve,  60 

Sarcolactic  acid,  39,  51 
Sarcolemma,  4 


Sarcosine,  44 

Schmidt,  A.,  on  tetanised  muscle,  52 

Schmidt,  Carl,  on  cerebro-spinal  fluid, 

74 

Schondorff,  on  urea  in  muscle  of 
mammals,  42 

Schroeder,  on  urea,  42 

Sclerosis,  disseminated,  129,  133 

Sciatic  nerves  of  cat,  137 

Serum  albumin,  22 

Serum  globulin,  22 

Sherrington,  on  stimulus  on  regenera- 
tion, 147 

Siegfried,  on  carnic  acid,  46,  50  ;  on 
retiform  tissue,  54 

Silver-chrome  process,  87 

Simin,  on  heat  rigor  of  heart  muscle, 
20 

Sleep,  96 

Solids,  of  nervous  tissues,  60  ;  organic, 
74,  75  ;  and  water,  relation  of,  57 

Soluble  myosin,  10 

Sowton,  Miss,  on  fatigue,  91,  95 

Soxhlet's  apparatus,  141 

Specific  gravity,  58 

Spermine,  128 

Sphygmometer  records  in  General 
Paralysis,  126 

Spina  bifida  cases,  fluid  from,  75 

Spinal  cord  haemorrhage,  148 

Splenic  nerves,  91,  94,  95 

Stadeler,  on  urea,  42 

Staircase  phenomenon,  83 

Starch,  21 

Stearic  acid,  66 

Stereo-chemical  isomerides,  37 

Stewart,  Purves,  on  nerve  regenera- 
tion, 144 

Stewart  and  Sollmann,  on  muscle  pro- 
teids, 9 

Steyrer,  on  muscle,  1 1 

Stimulation  fatigue,  95 

Sugar,  32,  35  ;  in  muscle,  35 

TAURINE,  32,  40 


i6o 


INDEX 


Tebb,  Miss,  globulins  and  albumin, 
22,  23,  25  ;  reticulin  in  tendon,  55  ; 
on  kephalin,  69 

Temperature,  coagulation,  16  ;  of  cell- 
globulin,  107  ;  influence  of,  on  the 
galvanometric  response  of  nerve  to 
stimulation,  102 

Temperature,  high  body,  20 

Tendo-mucoid,  53 

Tendon,  chemistry  of,  52 

Thesen,  on  iso-creatinine,  44 

Thierfelder,  on  cerebron,  70 

Thomson,  St  Clair,  on  cerebro-spinal 
fluid,  70,  71 

Thudichum,  on  phosphorised  fats  in 
brain,  68  ;  on  phrenosin  and  kerasin, 
69  ;  on  the  lability  of  lecithin,  83 

Tissue,  brain,  58  ;  retiform,  54 

Tissues,  chemical  composition  of 
nervous,  57  ;  contractile,  48  ;  con- 
nective, 52  ;  metabolism  in  nervous, 
78-85  ;  proteids  of  nervous,  61-63  ; 
reaction  of  nervous,  81  ;  solids  of 
nervous,  60 

Tonus,  chemical,  49 

Training  and  athletics,  34 

Trimethylamine,  127 

Trioxypurine,  45 

Trypsin,  12 

UREA,  32  ;  in  muscle,  41 
Uric  acid,  32,  44 
Urine,  proteids  in,  21 

VARIATION,  negative,   102  ;  diphasic, 

103 
Vaso  -  constrictor,     apparatus,     90  ; 

nerves,  94 


Velichi  on 

Vernon,  on  coagulation 

109 
Verworn,    Max,    on    carbon    dioxide, 

QO 
oo 

Vincent,  Swale,  on  muscle  proteids,  9  ; 

metabolism  in  nervous  tissues,  85 
Voit,  on  creatine,  43 
Voluntary  muscles,  3,  9 

WALLER,  on  carbon  dioxide,  82  ;  his 
dynamograph,  88 ;  on  the  axis- 
cylinder,  90  ;  his  hypothesis  of 
fatigue,  90,  95 

Wallerian  degeneration,  99,  134,  139 

Warm-blooded  and  cold-blooded  ani- 
mals compared  in  relation  to  muscle, 
14,  20;  to  nerve,  106,  107,  no,  114 

Warrington,  on  nerve-roots  and  -cells, 
149 

Water  and  solids,  relation  in  muscle, 
3  j  in  nerve,  57 

Weidel,  on  carnine,  41 

Weyl  and  Seitler,  on  acid  products  in 
muscle,  39 

Whartonian  jelly,  53 

Whitfield,  on  muscle,  9 

Woerner,  on  cerebron,  70 

Wright,  Hamilton,  on  narcosis,  98,  99, 
101 

XANTHINE,  32,  44,  63 
Xantho-creatinine,  43 

YOUNG,  R.  A.,  on  connective  tissues, 
52  ;  on  retiform  tissue,  55 

ZANIER,  on  cerebro-spinal  fluid,  77 


OF  THf 

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